Predictive biomarkers and use thereof to treat parkinson's disease

Abstract

The invention provides methods of treating patients with Parkinson's disease (PD) associated with LRRK2. The invention recognizes that analysis of biomarkers in such patients allows identification of those patients who will respond to LRRK2 inhibitors. Thus, the invention provides methods of identifying PD patients who will respond to LRRK2 inhibitors and methods of treating such patients.

Description

[0001] PREDICTIVE BIOMARKERS AND USE THEREOF TO TREAT PARKINSON’S DISEASE

[0002] Field of the Invention

[0003] The invention relates to methods of treating and diagnosing patients with Parkinson’s disease associated with leucine rich repeat kinase (LRRK2).

[0004] Background

[0005] Parkinson’s disease (PD) is a progressive neurodegenerative disease that affects over six million people globally. PD is usually recognized initially by motor impairment, with the cardinal symptoms being tremor, rigidity, slowness of movement, and difficulty with walking. In later stages, PD also produces neuropsychiatric disorders, including dementia, depression, and anxiety. PD afflicts more than 1% of people over the age of 60 and results in more than 100,000 deaths per year.

[0006] PD is thought to result from a confluence of genetic and environmental factors. Numerous mutations associated with familial PD have been identified, but 85-90% of PD cases are idiopathic. In PD cases that can be linked to known genetic factors, mutations in the LRRK2 gene are the most common cause of both familial and idiopathic PD. LRRK2 encodes a protein kinase that is expressed in multiple tissues including regions of the brain associated with PD such as the basal ganglia, and disease-causing mutations result in enhanced kinase activity. However, recent evidence indicates that some cases of PD are associated with increased activity of LRRK2.

[0007] Because no cure for PD exists, current treatments focus on alleviating symptoms, particularly motor impairment. The predominant approach for decades has been to enhance dopaminergic function using the dopamine precursor levodopa, a dopamine agonist, or a monoamine oxidase inhibitor. However, such medications lose their effectiveness as the disease progresses, and eventually their side effects outweigh their benefits.

[0008] Summary

[0009] More recently, the use of LRRK2 inhibitors has been investigated for treatment of PD cases associated with certain forms of the LRRK2 kinase. In the vast majority of PD cases, however, no rare variant in LRRK2 can be identified. Unfortunately, for PD patients with LRRK2 associated PD, there is no way to identify the subset of patients whose disease is associated with elevated LRRK2 activity, and LRRK2 inhibitors cannot be given to PD patients indiscriminately due to the risk of harm to patients who do not have pathological LRRK2 activity. Consequently, existing treatments for most PD patients are inadequate, and millions of people continue to suffer from the progressive and debilitating effects of the disease.

[0010] The invention solves this problem by using biomarkers to determine whether a PD patient has PD associated with LRRK2 and whether a patient would likely benefit from LRRK2 inhibitor therapy. The invention provides methods of determining whether a PD patient has PD associated with LRRK2 will be more likely to respond to a LRRK2 inhibitor using predictive biomarkers. The invention recognizes that biomarkers, including the ratio of glucosylceramide (GlcCer) to ceramide (Cer) may be relied upon as biomarkers to determine if LRRK2 inhibitor therapy would be effective for treatment of PD. The invention recognizes that the ratio of GlcCer to Cer may be an indicator for levels and activity of the LRRK2 kinase. Thus, the invention recognizes that the ratio of GlcCer to Cer serves as indicators to determine whether LRRK2 inhibitor therapy is appropriate for a given individual. Methods of the invention are useful both for identifying PD patients as candidates for LRRK2 inhibitor therapy and for treating such patients.

[0011] In one aspect, the invention provides a method of treating a patient having Parkinson’s disease associated with LRRK2, the method comprising: providing one or more LRRK2 inhibitors to the patient that presents with PD that is associated with LRRK2 and has an elevated ratio of GlcCer to Cer as compared to a subject not suffering from the neurological disease and has a PD associated with LRRK2, thereby treating PD associated LRRK2.

[0012] In certain embodiments, the ceramide species may be selected from the group consisting of (i) Cer (d 18: 1 / 16:0), (ii) Cer (d 18: 1 / 18:0), (iii) Cer (dl8:l / 24:0), (iv) Cer (dl8:l / 24:l), or a combination thereof. The GlcCer species may be selected from the group consisting of (i) GlcCer (d 18:1, 16:0), (ii) GlcCer (d 18: 1, 18:0), (iii) GlcCer (d 18: 1, 24:0), (iv) GlcCer (d 18:1, 24:1), or a combination thereof.

[0013] The ratio of GlcCer to Cer may be measured in any biofluid from the patient. In certain embodiments, the biofluid for measuring the ratio of GlcCer to Cer is urine, blood, cerebrospinal fluid (CSF), bile, or saliva. In certain preferred embodiments, the biofluid used for measuring the ratio of GlcCer to Cer is urine or CSF. In certain embodiments, the elevated ratio of GlcCer to Cer in the patient is at a concentration which indicates that the patient would be responsive to the one or more LRRK2 inhibitors.

[0014] In certain embodiments, the one or more LRRK2 inhibitors for therapy is selected from a group consisting of: CZC-25146, CZC-54252, DNL151, DNL201, GNE-7915, GSK2578215A, HG-10-102-01, JH-II-127, K252A, K252B, LRRK2-IN-1, MLi-2, PF-06447475, and staurosporine. In certain embodiments, the LRRK2 inhibitors are selected from a group consisting of: formulas (I), (II), (III), and (IV): wherein:

[0015] A is NH, O, S, C=O, NR3or CR4R5;

[0016] X is an optionally substituted arylene, heteroarylene, cycloalkylene, heterocycloalkylene, alkylcycloalkylene, heteroalkylcycloalkylene, aralkylene or heteroaralkylene group;

[0017] R1is an optionally substituted alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0018] R2is a hydrogen atom, a halogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0019] R3is an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkyl- cycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group; R4is a hydrogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group; and

[0020] R5is a hydrogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0021] B is NH, O, S, C=O, NR14or CR15R16;

[0022] R11is an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0023] R12is an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group, wherein R12is bound to the pyrimidine ring of formula (II) via a carbon-carbon bond;

[0024] R13is a hydrogen atom, a halogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0025] R14is an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkyl- cycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0026] R15is a hydrogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0027] R16is a hydrogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0028] R21is aryl or heteroaryl, each of which is optionally substituted;

[0029] R22is H, halo, OH, CN, CF3, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, C1-6 thioalkyl, C3-8 cycloalkyl, C2-8 heterocycloalkyl, aryl, or heteroaryl; and

[0030] Y is aryl or 5- or 6-membered heteroaryl; wherein each of the Ci-6 alkyl, C1-6 alkoxy, Ci- 6 haloalkyl, Ci-6 thioalkyl, C3-8 cycloalkyl, C2-8 heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one or more moieties selected from the group consisting of halo, OH, CN, CF3, NH2, NO2, C1-6 alkyl, C1-6 haloalkyl, C1-6 thioalkyl, C3-8 cycloalkyl, C2-8 heterocycloalkyl, C2-8 heterocycloalkenyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy, C1-6 haloalkoxy, Ci-6 alkylamino, C2-6 dialkylamino, C7-12 aralkyl, Ci-12 heteroaralkyl, aryl, heteroaryl, -C(O)R, -C(O)OR, -C(O)NRR’, -C(O)NRS(O)2R’, -C(O)NRS(O)2NR’R”, -OR, - OC(O)NRR’, -NRR’, -NRC(O)R’, -NRC(O)NR’R”, -NRS(O)2R’, -NRS(O)2NR’R”, -S(O)2R, and -S(O)2NRR’, in which each of R, R’, and R”, independently, is H, halo, OH, Ci-6 alkyl, Ci-6 haloalkyl, C1-6 alkoxy, C3-8 cycloalkyl, C2-8 heterocycloalkyl, aryl, or heteroaryl, or R and R’, or R’ and R”, together with the nitrogen to which they are attached, form C2-8 heterocycloalkyl;

[0031] R31is C(O)CH2R33, optionally substituted cycloalkyl, optionally substituted cycloheteroalkyl, optionally substituted cycloalkenyl, optionally substituted cycloheteroalkenyl, optionally substituted aryl, or optionally substituted heteroaryl; each instance of R32is independently halo, haloalkyl, optionally substituted alkoxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl;

[0032] R33is optionally substituted cycloalkyl, optionally substituted cycloheteroalkyl, optionally substituted cycloalkenyl, optionally substituted cycloheteroalkenyl, optionally substituted aryl, or optionally substituted heteroaryl;

[0033] Z is cycloalkyl, cycloheteroalkyl, cycloalkenyl, cycloheteroalkenyl, aryl, or heteroaryl; Z may be an aryl substituted with 2 or 3 instances of R2. Z may be a phenyl substituted with 2 or 3 instances of R2. Z may be a heteroaryl substituted with 2 or 3 instances of R2. Z may be a sixmembered heteroaryl substituted with 2 or 3 instances of R2; and n is 0-5, or a pharmaceutically acceptable salt of any compound described above.

[0034] In one aspect, the invention provides a compound of Formula (V):

[0035] or an enantiomer, mixture of enantiomers, tautomer, or pharmaceutically acceptable salt thereof, wherein: n is 1, 2, or 3;

[0036] Yi and Y2 are independently N or C;

[0037] Zi, Z2, and Z3 are independently selected from H, -OH, halo, cyano, amino, Ci-Ce alkyl, C1-C6 heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxy alkoxy;

[0038] X is H, halo, cyano, Ci-Ce alkyl, optionally deuterated Ci-Ce alkyl, C3-C6 cycloalkyl, Ci- Ce heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxy alkoxy;

[0039] Ri, R2, and R4 are independently selected from H, halo, cyano, Ci-Ce alkyl, optionally deuterated Ci-Ce alkyl, Ci-Ce heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxyalkoxy, with the proviso that these substitutions are permitted by valency;

[0040] W is H or C1-C4 substituted or unsubstituted alkyl, wherein W may optionally form a ring with Y2 when Y2 is C;

[0041] L is a linker, wherein L is a single bond, substituted or unsubstituted C 1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, wherein the one or more heteroatoms are selected from O, S, or N; A is 4-8 membered substituted or unsubstituted heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, wherein one or more heteroatoms are selected from a group consisting of O, S, orN; and wherein the substituents may be selected from a group consisting of substituted or unsubstituted 3-7 membered heterocycle, C1-C4 alkyl, -CH2-cycloalkyl, -CF2-cycloalky, -C(=O)- O-alkyl, halo, deuterium, cyano, cyanoalkyl, -CF3, mono-, di-, or tri-halo alkyl, CH(CH3)- cycloalkyl, -CH2-aryl, -CF2-aryl, -CH(-CH3)-aiyl, C(=O)-alkyl, -C(=O)cycloalkyl, -C(=O)-NH- alkyl, -COOH (and esters and carboxamides thereof), -C(=O)-morpholine, -C(=O)-heterocycles, -C(-CH3)2-OH, -CH2-C(=O)-NH2; -hydroxy, alkylhydroxy, alkyl-COOH (and esters and carboxamides thereof), amino, -NHC(=O)alkyl, -N(alkyl)C(=O)alkyl, -NHC(=O)aryl, - N(alkyl)C(=O)aryl, substituted or unsubstituted morpholine, 3-7 membered heterocycle, any of which may have one or more substituents, 3-7 membered cycloalkyl or heterocycle, wherein the 3-7 membered cycloalkyl or heterocycle is optionally fused with another 3-7 cycloalkyl or heterocycle, wherein the rings are spiro, bridged bicyclic, or spiro, wherein the at least one heteroatom in the heterocycle rings are independently selected from O, S, and N; and wherein one or more hydrogen atoms are optionally deuterium.

[0042] In another embodiment, in the compound of Formula (V), Yi is N and Y2 is C.

[0043] In another embodiment, in the compound of Formula (V), Yi is C and Y2 is N.

[0044] In another embodiment, in the compound of Formula (V), Yi is C and Y2 is C.

[0045] In another embodiment, in the compound of Formula (V), X is selected from a group consisting of -CH3, -CH2-CH3, -CD3, H, and F.

[0046] In another embodiment, in the compound of Formula (V), Zi, Z2, and Z3 are independently selected from H, F, or Cl.

[0047] In another embodiment, in the compound of Formula (V), Zi is F and Z2 is F.

[0048] In another embodiment, Z in the compound of Formula (V), 3 is H.

[0049] In another embodiment, in the compound of Formula (V), Z3 is F.

[0050] In another embodiment, in the compound of Formula (V), Ri is selected from H, F, -CH3, -CH2-CH3, -CF3, or -CHF2.

[0051] In another embodiment, in the compound of Formula (V), R2 is selected from a group consisting of H or F.

[0052] In another embodiment, in the compound of Formula (V), R2 is F. In another embodiment, in the compound of Formula (V), R4 is H.

[0053] In another embodiment, in the compound of Formula (V), L is a single bond.

[0054] In another embodiment, in the compound of Formula (V), L is alkyl.

[0055] In another embodiment, in the compound of Formula (V), L is -C=C~. In another embodiment, in the compound of Formula (V), L is -NH-CH2-.

[0056] In another embodiment, in the compound of Formula (V), A is not substituted or unsubstituted 1,2,3,6-Tetrahydropyridin.

[0057] In another embodiment, in the compound of Formula (V), A is selected from:

[0058]

[0059] In another embodiment, the compound of Formula (V) is selected from:

[0060]

[0061] In one aspect, the invention provides a compound of Formula (VI): or an enantiomer, mixture of enantiomers, tautomer, or pharmaceutically acceptable salt thereof, wherein: n is 1, 2, or 3;

[0062] Y is N or CR2;

[0063] Zi, Z2, and Z3 are independently selected from H, -OH, halo, cyano, amino, Ci-Ce alkyl, C1-C6 heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxy alkoxy;

[0064] X is H, halo, cyano, cycloalkyl, Ci-Ce alkyl, optionally deuterated Ci-Ce alkyl, Ci-Ce heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxyalkoxy;

[0065] Ri, R2, and R4 are independently selected from H, halo, cyano, Ci-Ce alkyl, optionally deuterated Ci-Ce alkyl, Ci-Ce heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxy alkoxy;

[0066] L is a linker, wherein L is a single bond, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, wherein the one or more heteroatoms are selected from O, S, or N;

[0067] A is 4-8 membered substituted or unsubstituted heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, wherein one or more heteroatoms are selected from a group consisting of O, S, or N; and wherein the substituents may be selected from a group consisting of substituted or unsubstituted 3-7 membered heterocycle, =0, -CH2-cycloalkyl, -CF2-cycloalky, -C(=O)-O-alkyl, halo, deuterium, cyano, cyanoalkyl, -CF3, mono-, di-, or tri-halo alkyl, CH(CH3)-cycloalkyl, - CH2-aiyl, -CF2-aryl, -CH(-CH3)-aryl, C(=O)-alkyl, -C(=O)cycloalkyl, -C(=O)-NH-alkyl, - COOH (and esters and carboxamides thereof), -C(=O)-morpholine, -C(=O)-heterocycles, -C(- CH3)2-OH, -CH2-C(=O)-NH2; -hydroxy, alkylhydroxy, alkyl-COOH (and esters and carboxamides thereof), amino, NHC(=O)alkyl, -N(alkyl)C(=O)alkyl, -NHC(=O)aryl, - N(alkyl)C(=O)aryl, -N(H)S(=O)2alkyl, -N(H)S(=O)2aryl, -N(alkyl)S(=O)2alkyl, cyanoalkyl, haloalkyl, -S=(0)2-alkyl, -S=(O)2-cycloalkyl, -S=(0)2-aryl, -S(=0)2N(H)alkyl, substituted or unsubstituted morpholine, 3-7 membered heterocycle, any of which may have one or more substituents, 3-7 membered cycloalkyl or heterocycle, wherein the 3-7 membered cycloalkyl or heterocycle is optionally fused with another 3-7 cycloalkyl or heterocycle, wherein the rings are spiro, bridged bicyclic, or spiro, wherein the at least one heteroatom in the heterocycle rings are independently selected from O, S, and N; and wherein one or more hydrogen atoms are optionally deuterium.

[0068] In another embodiment, in compounds of Formula (VI), Y is N.

[0069] In another embodiment, in compounds of Formula (VI), Y is CR2.

[0070] In another embodiment, in compounds of Formula (VI), Zi and Z2 are F.

[0071] In another embodiment, in compounds of Formula (VI), Zi and Z2 are independently selected from Cl or F.

[0072] In another embodiment, in compounds of Formula (VI), Z3 is H.

[0073] In another embodiment, in compounds of Formula (VI), Z3 is F.

[0074] In another embodiment, in compounds of Formula (VI), Z3 is -CH2-OH.

[0075] In another embodiment, in compounds of Formula (VI), X is selected from a group consisting of methyl, H, Cl, or -CD3.

[0076] In another embodiment, in compounds of Formula (VI), X is methyl.

[0077] In another embodiment, in compounds of Formula (VI), Ri is selected from H or methyl.

[0078] In another embodiment, in compounds of Formula (VI), R2 is H.

[0079] In another embodiment, in compounds of Formula (VI), R4 is H.

[0080] In another embodiment, in compounds of Formula (VI), L is a single bond.

[0081] In another embodiment, in compounds of Formula (VI), L is alkyl.

[0082] In another embodiment, in compounds of Formula (VI), L is -C=C-

[0083] In another embodiment, in compounds of Formula (VI), A is not substituted or unsubstituted 1,2,3,6-Tetrahydropyridin.

[0084] In another embodiment, in compounds of Formula (VI), A is morpholine with optional substitutions

[0085] In another embodiment, in compounds of Formula (VI), A is selected from: In another aspect, the invention provides a method of determining whether a patient having PD associated with LRRK2 will respond to a LRRK2 inhibitor, the method comprising: conducting an assay to measure the ratio of GlcCer to Cer from the patient; generating a report that identifies the ratio of GlcCer to Cer from the patient as compared to the ratio of GlcCer to Cer of a subject not suffering from PD and having PD associated with LRRK2; providing the report to a physician such that if the report indicates an elevated ratio of GlcCer to Cer in the patient as compared to the subject, the physician prescribes or provides the patient with one or more LRRK2 inhibitors.

[0086] In certain embodiments, the ceramide species may be selected from the group consisting of (i) Cer (d 18: 1 / 16:0), (ii) Cer (d 18: 1 / 18 :0), (iii) Cer (d 18:1 / 24:0), (iv) Cer (d 18: 1 / 24: 1), or a combination thereof. The GlcCer species may be selected from the group consisting of (i) GlcCer (d 18:1, 16:0), (ii) GlcCer (d 18: 1, 18:0), (iii) GlcCer (d 18: 1, 24:0), (iv) GlcCer (d 18:1, 24:1), or a combination thereof.

[0087] The ratio of GlcCer to Cer may be measured in any biofluid from the patient. In certain embodiments, the biofluid for measuring the ratio of GlcCer to Cer levels is urine, blood, cerebrospinal fluid (CSF), bile, or saliva. In certain preferred embodiments, the biofluid used for measuring the ratio of GlcCer to Cer is urine or CSF. In certain embodiments, the elevated ratio of GlcCer to Cer in the patient is at a concentration which indicates that the patient would be responsive to the one or more LRRK2 inhibitors. The LRRK2 inhibitor may be any of those described above.

[0088] In another aspect, the invention provides a method of treating a patient having PD associated withLRRK2, the method comprising: receiving data that identifies the ratio of GlcCer to Cer of the patient; comparing the data with the ratio of GlcCer to Cer from a subject not having PD and having PD associated with LRRK2; prescribing or providing the patient with one or more LRRK2 inhibitors if the patient has an elevated ratio of GlcCer to Cer as compared to the subject.

[0089] In certain embodiments, the ceramide species may be selected from the group consisting of (i) Cer (d 18 : 1 / 16:0), (ii) Cer (d 18: 1 / 18 :0), (iii) Cer (d 18 : 1 / 24:0), (iv) Cer (d 18: 1 / 24: 1), or a combination thereof. The GlcCer species may be selected from the group consisting of (i) GlcCer (d 18:1, 16:0), (ii) GlcCer (d 18: 1, 18:0), (iii) GlcCer (d 18: 1, 24:0), (iv) GlcCer (d 18:1, 24:1), or a combination thereof.

[0090] The ratio of GlcCer to Cer may be measured in any biofluid from the patient. In certain embodiments, the biofluid for measuring the ratio of GlcCer to Cer is urine, blood, cerebrospinal fluid (CSF), bile, or saliva. In certain preferred embodiments, the biofluid used for measuring ratio of GlcCer to Cer is urine or CSF. In certain embodiments, the elevated ratio of GlcCer to Cer in the patient are at a concentration which indicates that the patient would be responsive to the one or more LRRK2 inhibitors. The LRRK2 inhibitor may be any of those described above.

[0091] Brief Description of the Drawings

[0092] FIG. 1 is a graphs showing the ratio of GlcCer to Cer in different sets of patient populations.

[0093] FIG. 2A-2C are graphs showing the ratio of GlcCer to Cer in different sets patient populations.

[0094] Detailed Description

[0095] Parkinson’s disease (PD) is a progressive neurodegenerative disease that is caused by both genetic and environmental factors. One gene that plays a role in the development of some cases of PD is LRRK2, which encodes kinase that is expressed in multiple tissues including regions of the brain associated with PD such as the basal ganglia. Mutations in LRRK2 are the most common known genetic cause of PD, but patients with LRRK2 mutations make up a small fraction of the total number of PD cases. In particular, disease-causing mutations in LRRK2 result in increased activity of the LRRK2 kinase, and it has recently been shown that LRRK2 activity is elevated in some PD patients.

[0096] Various inhibitors of LRRK2 are currently being investigated as PD therapeutics. Such drugs hold promise for PD patients with LRRK2 mutations. However, the use of LRRK2 inhibitors to treat PD patients is problematic due to the varied etiology of the disease. Although patients with enhanced activity of LRRK2 would benefit from LRRK2 inhibitors, inhibition of LRRK2 may not be effective in PD patients who have normal levels of LRRK2 activity and whose disease pathology is attributable to changes in other molecular pathways. Because the neurons that express LRRK2 are located in the mid-brain and extremely difficult to access, activity of the kinase cannot be evaluated in living patients. Consequently, to date there has not been a means for identifying the subset of PD patients associated with LRRK2 that will benefit from LRRK2 inhibition.

[0097] The invention solves this problem by using biomarkers to determine whether a PD patient with PD associated with LRRK2 will likely benefit from a LRRK2 inhibitor. Consequently, methods of the invention allow candidates for LRRK2 pharmacotherapy to be identified based on genetic data that can be easily obtained from the patient. Thus, for a subset of PD patients, the invention unlocks the therapeutic potential of a class of drugs that were previously not recommended for them.

[0098] Parkinson’s disease and treatments thereof

[0099] Parkinson’s disease (PD) is a progressive neurodegenerative disease of the central nervous system. In early stages, the disease affects the motor system, and the cardinal symptoms are tremor, rigidity, slowness of movement, and difficulty with walking. Cognitive and behavioral symptoms, such as dementia, depression, and anxiety, often appear in later stages of PD. PD usually occurs in people over the age of 60, of whom about 1% are affected, but so- called early-onset PD may occur before the age of 50.

[0100] PD is characterized by the death of cells in the basal ganglia, including dopamine- secreting neurons, astrocytes, and microglia of the substantia nigra. Five mechanisms for neuronal death in PD have been proposed. First, the oligomerization of proteins, such as alpha- synuclein, into aggregates called Lewy bodies may lead directly to cell death. A second proposed cause is the dysregulation of autophagy, particularly degradation of mitochondria. Another proposed mechanism is that mitochondrial dysfunction leads to decreased energy production and an increase in reactive oxygen species. A fourth proposed mechanism is that due to neuroinflammation as a result of secretion of pro-inflammatory factors by the microglia. Finally, it has been proposed that breakdown of the blood -brain barrier allows plasma proteins to leak into the substantia nigra and promote apoptosis.

[0101] It is thought that PD results from a combination of genetic and environmental factors. In some cases, genetic mutations that increase the risk of PD are heritable, and about 10-15% of individuals with PD have a first-degree relative who has the disease. However, most instances of PD are idiopathic or “sporadic.” Genes with mutations that have been implicated in PD include CHCHD2, DJ1 / PARK7, DNAJC13, EIF4G1, GBA, LRRK2 / PARK8, PINK1, PRKN, SNCA, UCHL1, and VPS35. For both familial and sporadic PD, the most common known cause is mutation of LRRK2. Disease-causing mutations in LRRK2 result in a form of the kinase that has increased activity. Enhanced activity of LRRK2 has recently been implicated in idiopathic PD as well. The role of LRRK2 in PD is described in, for example, Chen, et al., Leucine-Rich Repeat Kinase 2 in Parkinson’s Disease: Updated from Pathogenesis to Potential Therapeutic Target, Eur Neurol. 2018;79(5-6):256-265, doi: 10.1159 / 000488938. Epub 2018 Apr 27; Di Maio, et al., LRRK2 activation in idiopathic Parkinson’s disease, Sci Transl Med. 2018 Jul 25;10(451):eaar5429, doi: 10.1126 / scitranslmed.aar5429; Taymans and Greggio, LRRK2 Kinase Inhibition as a Therapeutic Strategy for Parkinson’s Disease, Where Do We Stand? Curr Neuropharmacol. 2016;14(3):214-25, doi: 10.2174 / 1570159x13666151030102847, the contents of each of which are incorporated herein by reference.

[0102] Several behavioral and environmental conditions are known to increase the risk of developing PD. Risk factors associated with PD include exposure to pesticides and a history of head injury. Caffeine consumption and tobacco use are associated with decreased risk of PD. Low concentration of urate in the blood is associated with an increased risk of PD.

[0103] Management of PD usually entails pharmacological stimulation of the dopaminergic system. The most widely -used drug for treatment of PD is levodopa, which is enzymatically converted to dopamine in dopaminergic neurons. Dopamine agonists, such as bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, and lisuride, may also be used to treat PD. A third class of drugs for treatment of PD includes inhibitors of monoamine oxidase, such as selegiline and rasagiline.

[0104] Ceramide and glucosylceramide

[0105] Ceramides belong to the class of sphingolipids characterized by the presence of a sphingosine moiety and a fatty acid. In cell membranes, ceramides are essential structural elements of lipid bilayers. In the brain, ceramides play a variety of functions to coordinate brain homeostasis, and neuronal plasma membranes tend to be rich in ceramides. Aging brains display increased ceramide content and the accumulation of ceramide is associated with impaired receptor trafficking and synapse loss.

[0106] Glucosylceramide (GlcCer) is a ceramide also composed of a glucose moiety. GlcCer is found in plants, fungi, and animals and absent in bacteria and in some eukaryotes such as specific yeasts. Glucosylceramide is synthesized in the Golgi apparatus by glucosylceramide synthase via the transfer of a glucose residue from UDP-glucose to ceramide. It is found in all mammalian tissues, particularly abundant in the brain, and is required for intracellular membrane trafficking, signal activity, and cell proliferation.

[0107] Lipid buildup due to impaired lysosomal degradative capacity of neurons or glia cells has been known to result in lysosomal storage diseases (LSDs). Heterozygous mutations in glucocerebrosidase, a lysosomal enzyme breaking down glucosylceramide into glucose and ceramide, increases by five folds the lifetime risk of Parkinson’s disease (PD).

[0108] Ceramide may be of multiple species. A shorthand nomenclature combines the labeling used for fatty acids and long-chain bases to denote molecular species of ceramides, including those as components of more complex lipids. Using C18 Ceramide (d 18:1 / 18:0) as an example, C 18 represents the number of carbons in the fatty acid chain, Ceramide indicates the common family name, d indicates the number of -OH moieties, 18:1 indicates the length of the sphingoid backbone, and 18:0 indicates the fatty acid chain length and saturation. For example, N- palmitoyl-sphingosine is characterized by d 18:1 / 16:0. Common sphingoid bases are identified in the table below:

[0109] Ceramide species include (i) Cer (d 18 : 1 / 16:0), (ii) Cer (d 18:1 / 18:0), (iii) Cer (d 18: 1 / 24:0), (iv) Cer (d 18 : 1 / 24: 1 ), or a combination thereof. Very -long-chain ceramides containing 24:0 or 24:1 fatty acids turn over much more rapidly in animal cells than those containing 16:0 or 18:0 fatty acids. In contrast, ceramides containing d 16: 1 and d 18: 1 sphingoid bases turnover at comparable rates. The GlcCer may be of the species (i) GlcCer (d 18: 1, 16:0), (ii) GlcCer (d 18: 1, 18:0), (iii) GlcCer (d 18: 1, 24:0), (iv) GlcCer (d 18: 1, 24:1), or a combination thereof. Rare variants

[0110] LRRK2 is the most common genetic cause of Parkinson’s disease. Greater than 100 mutations in LRRK2 cause or contribute to Parkinson’s disease with a clear association between LRRK2 kinase activity and disease risk, also referred to as penetrance. Approximately 1 -3% of Parkinson’s disease patients in the US have a rare variant of LRRK2 with elevated LRRK2 kinase activity. Numerous SNPs upstream of LRRK2 are also known to alter the mRNA expression of LRRK2 in multiple tissues, including the brain.

[0111] Identification of genetic modifiers and rare variants

[0112] The invention provides methods of determining whether a PD patient has PD associated with LRRK2 and will be more likely to respond to a LRRK2 inhibitor using genetic modifiers of LRRK2 in the patient’s genome as indicators. The invention recognizes that genetic modifiers of LRRK2 may cause changes, e g., increases or decreases, in the level or activity of the LRRK2 kinase or may otherwise alter LRRK2 signaling pathways via upstream or downstream regulators and thus contribute to PD etiology. Consequently, PD patients who have one or more such modifiers may benefit from pharmacotherapy using a LRRK2 inhibitor despite having LRRK2 alleles that produce normal forms of the kinase. Thus, genetic modifiers of LRRK2 activity serve as indicators to determine whether LRRK2 inhibitor therapy is appropriate for a given individual. Methods of the invention are useful both for identifying PD patients as candidates for LRRK2 inhibitor therapy and for treating such patients.

[0113] In an aspect, the invention provides methods of treating a subject having Parkinson’s disease associated with LRRK2 by providing a LRRK2 inhibitor to a subject that presents with Parkinson’s disease and that has PD associated LRRK2 and a genetic modifier of LRRK2 such that the subject will respond to the LRRK2 inhibitor, thereby treating Parkinson’s disease associated with LRRK2 in the subject.

[0114] The genetic data may comprise any type of data on the composition and / or expression of one or more genes in the subject. The genetic data may include one or more of exomic, genomic, genotypic, proteomic, sequence, and transcriptomic data.

[0115] The genetic modifier may be any genetic element that modifies, or correlates with the change in activity of, LRRK2 expression or activity, or that causes a change in protein levels associated with disease burden (whether increased or reduced). The genetic modifier may increase or decrease expression and / or activity of LRRK2; the genetic modifier may also increase or reduce degradation of LRRK2. The genetic modifier may be an amplification, deletion, duplication, fusion, insertion, inversion, rearrangement, single nucleotide polymorphism (SNP), substitution, or translocation. The genetic modifier may lie within a coding region or a non-coding region in the subject’s genome. The genetic modifier may be associated with family history and genetically ascertained Ashkenazi status.

[0116] In certain embodiments, the genetic modifier may be any genetic modifier provided in PCT / US2021 / 056443, which is incorporated by reference in its entirety. In certain embodiments, the SNP may be any of the SNP listed in PCT / US2021 / 056443, which is incorporated by reference in its entirety.

[0117] The SNP may be rsl0784722, rs!0877877, rs!0879122, rsl 1181542, rsl 13111234, rsl 13736300, rsl2230765, rsl2816484, rsl2829831, rsl3377670, rsl41551396, rsl44377852, rsl49173058, rsl7580794, rsl7621741, rsl838354, rsl84120094, rsl88535877, rsl88583486, rsl88604552, rsl89517205, rs200611801, rs200907772, rs201889643, rs201944175, rs2406426, rs2406860, rs285561, rs34566033, rs368141132, rs369084695, rs371700002, rs371905892, rs373439540, rs376468815, rs377104202, rs377627337, rs384234, rs61920964, rs6581941, rs6650226, rs71078241, rs7304080, rs73088926, rs74434364, rs74842215, rs75043969, rs78468120, rs7960429, rs7979420, rs76904798, rs57025360, rsl 12515153, rsl0877877, rsl0784722, rs4272849, rs2404832, rsl 17534366, rsl838343, rsl0880342, rsl 1177660, rsl83028452, rsl l6912628, rsl47755361, rsl l584630, rs3793397, rsl l l794893, rs4931640, rs526507, rs79307177, rsl87116363, rs71609573, rs74390551, rsl44665441, rsl718880, rsl991401, rsl 1052225, rsl45801597, rs72907976, rsl47286120, rs378690, rs73188365, rs610037, rs75479531, rsl 112191556, rs3O83O3, rsl0790282, rs3729912, rs4326638, rs4414548, rsl3009437, rs56045011, rs6858566, rs4425, rsl 1052253, or any other SNPs in linkage disequilibrium (LD) with these SNPs that would be suitable as a proxy for these SNPs. The LRRK2 inhibitor may be any of the inhibitors listed in this application.

[0118] In certain embodiments, the SNP may be rs33939927, rs35801418, rs34805604, rs34637584, rs35870237, rs34995376, rs34778348, rsl 1611119, rs6581439, rsl2296462, rs549790, rsl2423473, rs555740, rs2242367, rs7295598, rs2253736, rsl 1564274, rs2708419, rsl7519419, rs76904798, rsl7519573, rsl l l75847, rsl0878452, rsl7444612, rsl l7929583, rsl 1564235, rsl7128233, rs7960976, rsl918942, rs611829, rs7531501, rs9793102, rs3755541, rs7578955, rsl7738103, rs4676776, rsl259475, rsl0477505, rs7380062, rs4636028, rsl2704998, rs2768282, rsl0283642, rs3750779, rsl362993, rs7089200, rs9783486, rs4763946, rsl6920645, rs!2312400, rs2708494, rsl2813279, rsl 1613339, rs7300813, rs!66806, rs3794253, rs7960429, rs7955116, rsl0491998, rsl2814145, rs2708078, rsl922761, rsl373422, rsl7691793, rs7301498, rs7967809, rsl866074, rsl0507535, rs9300705, rsl642819, rs8048361, rsl 152838, rs2331796, rsl0515969, rs6566942, or rs2422956.

[0119] In another aspect, the invention provides methods of determining whether a subject having Parkinson’s disease associated with LRRK2 will respond to a LRRK2 inhibitor. The methods includes conducting an assay on a sample from a subject that has Parkinson’s disease associated with LRRK2 in order to obtain genetic data from a subject, generating a report that identifies one or more genetic modifier of LRRK2 in the genetic data, wherein the one or more genetic modifiers in the LRRK2 network are indicative that the subject having Parkinson’s disease associated with LRRK2 will be responsive to a LRRK2 inhibitor, and providing the report to a physician such that the physician prescribe or provide the subject with a LRRK2 inhibitor.

[0120] The genetic data may be any type of genetic data described above. The genetic modifier may be any type of genetic modifier of LRRK2 described above. The genetic modifier may be any of the SNPs listed above. The LRRK2 inhibitor may be any of the inhibitors listed in this application.

[0121] In another aspect, the invention provides methods of treating a subject having PD associated with LRRK2. The methods include receiving genetic data that identifies one or more genetic modifier of LRRK2, wherein the one or more genetic modifiers are indicative that a subject having Parkinson’s disease associated with LRRK2 will be responsive to a LRRK2 inhibitor, and prescribing or providing the subject with a LRRK2 inhibitor. The genetic data may be any type of genetic data described above. The genetic modifier may be any type of genetic modifier of LRRK2 described above. The genetic modifier may be any of the SNPs listed above.

[0122] The invention also recognizes that genetic modifiers of LRRK2 serve as indicators that PD patients having LRRK2 are likely to benefit from pharmacotherapy using one or more LRRK2 inhibitors. A genetic modifier of LRRK2 may be one or more genetic elements (e.g., a single genetic element alone or any combination(s) of genetic elements) that operably modifies LRRK2, e.g., that alters the expression, degradation, localization (e.g., within a cell or across cell types), binding, or activity of LRRK2, including the LRRK2 gene, transcripts of the LRRK2 gene, and polypeptide products of the LRRK2 gene, in a subject. For example and without limitation, a genetic modifier may alter, e.g., increase or decrease, expression, activity, stability, binding, localization, degradation, transcription, or translation of LRRK2, including the LRRK2 gene, transcripts of the LRRK2 gene, and polypeptide products of the LRRK2 gene. In certain embodiments, a genetic modifier of LRRK2 may be a structural variation in the genome of the subject. For example and without limitation, a genetic modifier may be an amplification, deletion, duplication, fusion, insertion, inversion, rearrangement, single nucleotide polymorphism (SNP), substitution, or translocation. SNPs that may be genetic modifiers of LRRK2 are listed in Example 1. In addition, any other SNPs that are in linkage disequilibrium (LD) with the SNPs listed in Example 1 may be used as a genetic modifier. A genetic modifier may be a cis-regulatory element, such as a promoter, enhancer, silencer, or operator. The cis- regulatory element may regulate the binding of one or more proteins to DNA in proximity to LRRK2. The cis-regulatory element may affect binding of a histone, transcription factor, initiation factor, helicase, polymerase, or component of any of the aforementioned proteins. A genetic modifier may be a trans-acting factor. The trans-acting factor may affect transcription or translation of LRRK2. A genetic modifier may be in any region of the subject’s genome. A genetic modifier may lie within a coding region or non-coding region of the subject’s genome. The coding region may be in LRRK2 or in another gene. A genetic modifier may lie within the LRRK2 coding region but not alter the sequence of the LRRK2 polypeptide, the size of the LRRK2 polypeptide, or both.

[0123] Methods of the invention may include identification or analysis of one or more genetic modifiers of LRRK2 in genetic data obtained from a subject. The genetic data may comprise any type of data on the composition and / or expression of one or more genes in the subject. The genetic data may include one or more of exomi c, genomic, genotypic, proteomic, sequence, and transcriptomic data. The genetic data may include data on one or more genes known to be associated with PD, such as any of those described above.

[0124] Genetic modifiers may be identified from genetic data using any suitable method. In some embodiments, the genetic data collected from the subject is compared to a reference set of data in order to provide a probability of responsiveness to a LRRK2 inhibitor. The reference set may include data collected from individuals that do not have PD. Phenotypic data from subjects and reference individuals may also be used. Phenotypic data may contain traits associated with PD, including PD symptoms or PD risk factors, such as those described above. Data may include outcomes, such as whether the individual responded to LRRK2 inhibitor treatment.

[0125] The invention provides methods and systems for predicting a subject’s responsiveness to a LRRK2 inhibitor based on the subject’s phenotypic traits and / or genotypic data. In some embodiments, methods and systems of the invention use a diagnostic signature for predicting responsiveness. The diagnostic predictor can be based on any appropriate pattern recognition method that receives input data representative of a plurality of responsiveness -associated phenotypic traits, such as molecular signatures of (1) LRRK2-like manifestations of PD observed in carriers of LRRK2 deleterious variants, (2) PD of apparently unknown mechanism, and (3) appropriate controls, and provides an output that indicates a probability that the subject will respond to a LRRK2 inhibitor. The diagnostic predictor may be trained with data from a plurality of individuals for whom phenotypic traits, medical interventions, and LRRK2 inhibitor response outcomes are known. The plurality of individuals used to train the diagnostic predictor is also known as the training population. For each individual in the training population, the training data comprises (a) data representative of a plurality of phenotypic traits; (b) medical interventions; and (c) LRRK2 inhibitor response information. LRRK2 inhibitor response outcome may not be required to generate a diagnostic signature. LRRK2 inhibitor responses can be evaluated in a prospectively selected patient population. Various diagnostic predictors that can be used in conjunction with the present invention are described below. In some embodiments, additional individuals having known trait profiles and LRRK2 response outcomes can be used to test the accuracy of the diagnostic predictor obtained using the training population. Such additional patients are known as the testing population.

[0126] In certain embodiments, the methods of invention use a diagnostic predictor, also called a classifier, for determining the probability of responding to LRRK2 inhibition. As noted above, the diagnostic predictor can be based on any appropriate pattern recognition method that receives a profile, such as a profile based on a plurality of phenotypic traits and provides an output comprising data indicating a that a patient is more or less likely to respond to a LRRK2 inhibitor, and may include possible risks and benefits of treatment with such an inhibitor. The profile can be obtained by completion of a questionnaire containing questions regarding certain phenotypic traits or the collection of a biological sample to obtain genotypic data or a combination thereof. The diagnostic predictor is trained with training data from a training population of individuals for whom phenotypic traits, medical interventions, and LRRK2 inhibitor response outcomes are known.

[0127] A diagnostic predictor based on any of such methods can be constructed using the profiles and diagnostic data of the training patients. Such a diagnostic predictor can then be used to predict the LRRK2 inhibitor response of a subject based on her profile of phenotypic traits, genotypic traits, or both. The methods can also be used to identify traits that discriminate between responding and not responding to LRRK2 inhibition using a trait profile and diagnostic data of the training population.

[0128] In one embodiment, the diagnostic predictor can be prepared by (a) generating a reference set of individuals for whom phenotypic traits, medical interventions, and LRRK2 response outcomes are known; (b) determining for each trait, a metric of correlation between the trait and LRRK2 response outcome in a plurality of individuals having known LRRK2 response outcomes at a predetermined time; (c) selecting one or more traits based on said level of association; (d) training a diagnostic predictor, in which the diagnostic predictor receives data representative of the traits selected in the prior step and provides an output indicating a probability of responding to LRRK2 inhibition, with training data from the reference set of subjects including assessments of traits taken from the individuals.

[0129] Various known statistical pattern recognition methods can be used in conjunction with the present invention. The statistical pattern recognition methods are described in detail above.

[0130] Assays to obtain genetic data

[0131] The identification or analysis of one or more genetic modifiers of LRRK2 may include performing an assay on a sample obtained from a subject. The sample may be any type of sample that contains genetic material, such as DNA or RNA. For example and without limitation, the sample may be from an amniotic fluid, biopsy, blood, bodily fluid, cell, cerebrospinal fluid, lymphatic fluid, mouthwash, needle aspiration biopsy, hair, phlegm, plasma, pus, saliva, semen, serum, sputum, stool, swab, sweat, synovial fluid, tear, tissue, urine, or a combination of any of the aforementioned samples. For example and without limitation, a tissue sample may be from bone marrow tissue, CNS tissue, eye tissue, gastrointestinal tissue, genitourinary tissue, hair, kidney tissue, liver tissue, mammary gland tissue, mammary gland tissue, musculoskeletal tissue, nails, nasal passage tissue, neural tissue, placental tissue, placental tissue, or skin tissue. The subject may be any type of subject. The subject may be a human. The subject may show one or more symptoms of Parkinson’s disease, or the subject may be asymptomatic. The patient may be related to a PD patient. The subject may be a pediatric patient, a newborn, a neonate, an infant, a child, an adolescent, a pre-teen, a teenager, an adult, or an elderly subject. The subject may show one or more symptoms of Parkinson’s disease, or the subject may be asymptomatic. The patient may be related to a PD patient.

[0132] Methods of genetic analysis are known in the art. In certain embodiments, a known single nucleotide polymorphism at a particular position can be detected by single base extension for a primer that binds to the sample DNA adjacent to that position, as described in, for example, U.S. Patent No. 6,566,101, the content of which is incorporated by reference herein in its entirety. In other embodiments, a hybridization probe might be employed that overlaps the SNP of interest and selectively hybridizes to sample nucleic acids containing a particular nucleotide at that position, as described in, for example, U.S. Patent Nos. 6,214,558 and 6,300,077, the contents of which are incorporated by reference herein in their entirety.

[0133] In particular embodiments, nucleic acids are sequenced in order to detect variants (i.e., mutations) in the nucleic acid compared to various forms of the sequence. The nucleic acid can include a plurality of nucleic acids derived from a plurality of genetic elements. Methods of detecting sequence variants are known in the art, and sequence variants can be detected by any sequencing method known in the art e.g., ensemble sequencing or single molecule sequencing.

[0134] Sequencing may be by any method known in the art. DNA sequencing techniques include classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, allele specific hybridization to a library of labeled oligonucleotide probes, sequencing by synthesis using allele specific hybridization to a library of labeled clones that is followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, polony sequencing, and SOLiD sequencing. Sequencing of separated molecules has more recently been demonstrated by sequential or single extension reactions using polymerases or ligases as well as by single or sequential differential hybridizations with libraries of probes

[0135] One conventional method to perform sequencing is by chain termination and gel separation, as described in, for example, Sanger et al., Proc Natl. Acad. Sci. U S A, 74(12): 5463 67 (1977). Another conventional sequencing method involves chemical degradation of nucleic acid fragments, as described in, for example, Maxam et al., Proc. Natl. Acad. Sci., 74: 560 564 (1977). Finally, methods have been developed based upon sequencing by hybridization, as described in, for example, U.S. Patent Publication number 2009 / 0156412. The content of each reference is incorporated by reference herein in its entirety.

[0136] A sequencing technique that can be used in the methods of the provided invention includes, for example, Harris T. D. et al., Single-Molecule DNA Sequencing of a Viral Genome, (2008) Science 320:106-109. In the true single molecule sequencing (tSMS) technique, a DNA sample is cleaved into strands of approximately 100 to 200 nucleotides, and a polyA sequence is added to the 3' end of each DNA strand. Each strand is labeled by the addition of a fluorescently labeled adenosine nucleotide. The DNA strands are then hybridized to a flow cell, which contains millions of oligo-T capture sites that are immobilized to the flow cell surface. The templates can be at a density of about 100 million templates / cm2. The flow cell is then loaded into an instrument, e.g., HeliScope.TM. sequencer, and a laser illuminates the surface of the flow cell, revealing the position of each template. A CCD camera can map the position of the templates on the flow cell surface. The template fluorescent label is then cleaved and washed away. The sequencing reaction begins by introducing a DNA polymerase and a fluorescently labeled nucleotide. The oligo-T nucleic acid serves as a primer. The polymerase incorporates the labeled nucleotides to the primer in a template directed manner. The polymerase and unincorporated nucleotides are removed. The templates that have directed incorporation of the fluorescently labeled nucleotide are detected by imaging the flow cell surface. After imaging, a cleavage step removes the fluorescent label, and the process is repeated with other fluorescently labeled nucleotides until the desired read length is achieved. Sequence information is collected with each nucleotide addition step. Further description of tSMS is shown for example in U.S. Patent Nos. 7,169,560; 6,818,395; and 7,282,337; U.S. Patent Publication Nos. 2009 / 0191565 and 2002 / 0164629; and Braslavsky, et al., PNAS (USA), 100: 3960-3964 (2003), the contents of each of which are incorporated by reference herein in their entirety.

[0137] Another example of a DNA sequencing technique that can be used in the methods of the provided invention is 454 sequencing (Roche), as described in, for example, Margulies, M et al. 2005, Nature, 437, 376-380. 454 sequencing involves two steps. In the first step, DNA is sheared into fragments of approximately 300-800 base pairs, and the fragments are blunt ended. Oligonucleotide adaptors are then ligated to the ends of the fragments. The adaptors serve as primers for amplification and sequencing of the fragments. The fragments can be attached to DNA capture beads, e.g., streptavidin-coated beads using, e.g., Adaptor B, which contains 5'- biotin tag. The fragments attached to the beads are PCR amplified within droplets of an oil-water emulsion. The result is multiple copies of clonally amplified DNA fragments on each bead. In the second step, the beads are captured in wells (pico-liter sized). Pyrosequencing is performed on each DNA fragment in parallel. Addition of one or more nucleotides generates a light signal that is recorded by a CCD camera in a sequencing instrument. The signal strength is proportional to the number of nucleotides incorporated. Pyrosequencing makes use of pyrophosphate (PPi) which is released upon nucleotide addition. PPi is converted to ATP by ATP sulfurylase in the presence of adenosine 5' phosphosulfate. Luciferase uses ATP to convert luciferin to oxyluciferin, and this reaction generates light that is detected and analyzed.

[0138] Another example of a DNA sequencing technique that can be used in the methods of the provided invention is SOLiD technology (Applied Biosystems). In SOLiD sequencing, genomic DNA is sheared into fragments, and adaptors are attached to the 5' and 3' ends of the fragments to generate a fragment library. Alternatively, internal adaptors can be introduced by ligating adaptors to the 5' and 3' ends of the fragments, circularizing the fragments, digesting the circularized fragment to generate an internal adaptor, and attaching adaptors to the 5' and 3' ends of the resulting fragments to generate a mate-paired library. Next, clonal bead populations are prepared in microreactors containing beads, primers, template, and PCR components. Following PCR, the templates are denatured, and beads are enriched to separate the beads with extended templates. Templates on the selected beads are subjected to a 3' modification that permits bonding to a glass slide. The sequence can be determined by sequential hybridization and ligation of partially random oligonucleotides with a central determined base (or pair of bases) that is identified by a specific fluorophore. After a color is recorded, the ligated oligonucleotide is cleaved and removed, and the process is then repeated.

[0139] Another example of a DNA sequencing technique that can be used in the methods of the provided invention is Ion Torrent sequencing, as described in U.S. Patent Publication Nos. 2009 / 0026082, 2009 / 0127589, 2010 / 0035252, 2010 / 0137143, 2010 / 0188073, 2010 / 0197507, 2010 / 0282617, 2010 / 0300559, 2010 / 0300895, 2010 / 0301398, and 2010 / 0304982, the contents of each of which are incorporated by reference herein in their entirety. In Ion Torrent sequencing, DNA is sheared into fragments of approximately 300-800 base pairs, and the fragments are blunt ended. Oligonucleotide adaptors are then ligated to the ends of the fragments. The adaptors serve as primers for amplification and sequencing of the fragments. The fragments can be attached to a surface and is attached at a resolution such that the fragments are individually resolvable. Addition of one or more nucleotides releases a proton (H+), which signal detected and recorded in a sequencing instrument. The signal strength is proportional to the number of nucleotides incorporated.

[0140] Another example of a sequencing technology that can be used in the methods of the provided invention is Illumina sequencing. Illumina sequencing is based on the amplification of DNA on a solid surface using fold-back PCR and anchored primers. Genomic DNA is fragmented, and adapters are added to the 5' and 3' ends of the fragments. DNA fragments that are attached to the surface of flow cell channels are extended and bridge amplified. The fragments become double stranded, and the double stranded molecules are denatured. Multiple cycles of the solid -phase amplification followed by denaturation can create several million clusters of approximately 1,000 copies of single-stranded DNA molecules of the same template in each channel of the flow cell. Primers, DNA polymerase and four fluorophore -labeled, reversibly terminating nucleotides are used to perform sequential sequencing. After nucleotide incorporation, a laser is used to excite the fluorophores, and an image is captured, and the identity of the first base is recorded. The 3' terminators and fluorophores from each incorporated base are removed and the incorporation, detection and identification steps are repeated.

[0141] Another example of a sequencing technology that can be used in the methods of the provided invention includes the single molecule, real-time (SMRT) technology of Pacific Biosciences. In SMRT, each of the four DNA bases is attached to one of four different fluorescent dyes. These dyes are phospholinked. A single DNA polymerase is immobilized with a single molecule of template single stranded DNA at the bottom of a zero-mode waveguide (ZMW). A ZMW is a confinement structure which enables observation of incorporation of a single nucleotide by DNA polymerase against the background of fluorescent nucleotides that rapidly diffuse in an out of the ZMW (in microseconds). It takes several milliseconds to incorporate a nucleotide into a growing strand. During this time, the fluorescent label is excited and produces a fluorescent signal, and the fluorescent tag is cleaved off. Detection of the corresponding fluorescence of the dye indicates which base was incorporated. The process is repeated.

[0142] Another example of a sequencing technique that can be used in the methods of the provided invention is nanopore sequencing, as described in, for example, Soni G V and Meller A. (2007) Clin Chem 53: 1996-2001. A nanopore is a small hole, of the order of 1 nanometer in diameter. Immersion of a nanopore in a conducting fluid and application of a potential across it results in a slight electrical current due to conduction of ions through the nanopore. The amount of current which flows is sensitive to the size of the nanopore. As a DNA molecule passes through a nanopore, each nucleotide on the DNA molecule obstructs the nanopore to a different degree. Thus, the change in the current passing through the nanopore as the DNA molecule passes through the nanopore represents a reading of the DNA sequence.

[0143] Another example of a sequencing technique that can be used in the methods of the provided invention involves using a chemical-sensitive field effect transistor (chemFET) array to sequence DNA, for example, as described in U.S. Patent Publication No. 20090026082). In one example of the technique, DNA molecules can be placed into reaction chambers, and the template molecules can be hybridized to a sequencing primer bound to a polymerase. Incorporation of one or more triphosphates into a new nucleic acid strand at the 3' end of the sequencing primer can be detected by a change in current by a chemFET. An array can have multiple chemFET sensors. In another example, single nucleic acids can be attached to beads, and the nucleic acids can be amplified on the bead, and the individual beads can be transferred to individual reaction chambers on a chemFET array, with each chamber having a chemFET sensor, and the nucleic acids can be sequenced.

[0144] Another example of a sequencing technique that can be used in the methods of the provided invention involves using an electron microscope, as described in, for example, Moudrianakis E. N. and Beer M. Proc Natl Acad Sci USA. 1965 March; 53:564-71. In one example of the technique, individual DNA molecules are labeled using metallic labels that are distinguishable using an electron microscope. These molecules are then stretched on a flat surface and imaged using an electron microscope to measure sequences.

[0145] If the nucleic acid from the sample is degraded or only a minimal amount of nucleic acid can be obtained from the sample, PCR can be performed on the nucleic acid in order to obtain a sufficient amount of nucleic acid for sequencing, as described in, for example, U.S. Patent No. 4,683,195, the content of which is incorporated by reference herein in its entirety).

[0146] Methods of detecting levels of gene products (e.g., RNA or protein) are known in the art. Commonly used methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization, as described in, for example, Parker & Bames, Methods in Molecular Biology 106:247-283 (1999), the contents of which are incorporated by reference herein in their entirety; RNAse protection assays, Hod, Biotechniques 13:852 854 (1992), the contents of which are incorporated by reference herein in their entirety); and PCR-based methods, such as reverse transcription polymerase chain reaction (RT-PCR), Weis et al., Trends in Genetics 8:263 264 (1992), the contents of which are incorporated by reference herein in their entirety. Alternatively, antibodies may be employed that can recognize specific duplexes, including RNA duplexes, DNA-RNA hybrid duplexes, or DNA-protein duplexes. Other methods known in the art for measuring gene expression (e.g., RNA or protein amounts) are shown in, for example, U.S. Patent Publication No. 2006 / 0195269, the content of which is hereby incorporated by reference in its entirety.

[0147] A differentially or abnormally expressed gene refers to a gene whose expression is activated to a higher or lower level in a subject suffering from a disorder, such as PD, relative to its expression in a normal or control subject. The terms also include genes whose expression is activated to a higher or lower level at different stages of the same disorder. It is also understood that a differentially expressed gene may be either activated or inhibited at the nucleic acid level or protein level, or may be subject to alternative splicing to result in a different polypeptide product. Such differences may be evidenced by a change in mRNA levels, surface expression, secretion or other partitioning of a polypeptide, for example.

[0148] Differential gene expression may include a comparison of expression between two or more genes or their gene products, or a comparison of the ratios of the expression between two or more genes or their gene products, or even a comparison of two differently processed products of the same gene, which differ between normal subjects and subjects suffering from a disorder, such as PD, or between various stages of the same disorder. Differential expression includes both quantitative, as well as qualitative, differences in the temporal or cellular expression pattern in a gene or its expression products. Differential gene expression (increases and decreases in expression) is based upon percent or fold changes over expression in normal cells. Increases may be of 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200% relative to expression levels in normal cells. Alternatively, fold increases may be of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold over expression levels in normal cells. Decreases may be of 1, 5, 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% relative to expression levels in normal cells.

[0149] In certain embodiments, reverse transcriptase PCR (RT-PCR) is used to measure gene expression. RT-PCR is a quantitative method that can be used to compare mRNA levels in different sample populations to characterize patterns of gene expression, to discriminate between closely related mRNAs, and to analyze RNA structure.

[0150] The first step is the isolation of mRNA from a target sample. The starting material is typically total RNA isolated from human tissues or fluids.

[0151] General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987), and De Andres et al., BioTechniques 18:42044 (1995). The contents of each of these references are incorporated by reference herein in their entirety. In particular, RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Other commercially available RNA isolation kits include MASTERPURE Complete DNA and RNA Purification Kit (EPICENTRE, Madison, Wis.), and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumor can be isolated, for example, by cesium chloride density gradient centrifugation.

[0152] The first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction. The two most commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo -dT primers, depending on the circumstances and the goal of expression profiling. For example, extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, Calif, USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction.

[0153] Although the PCR step can use a variety of thermostable DNA-dependent DNA polymerases, it typically employs the Taq DNA polymerase, which has a 5'-3' nuclease activity but lacks a 3'-5' proofreading endonuclease activity. Thus, TAQMAN PCR typically utilizes the 5'-nuclease activity of Taq polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used. Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.

[0154] TAQMAN RT-PCR can be performed using commercially available equipment, such as, for example, ABI PRISM 7700TM Sequence Detection SystemTM (Perkin -Elmer-Applied Biosystems, Foster City, Calif., USA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In certain embodiments, the 5' nuclease procedure is run on a real-time quantitative PCR device such as the ABI PRISM 7700TM Sequence Detection System TM. The system consists of a thermocycler, laser, charge -coupled device (CCD), camera and computer. The system amplifies samples in a 96-well format on a thermocycler. During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data.

[0155] 5'-Nuclease assay data are initially expressed as Ct, or the threshold cycle. As discussed above, fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction. The point when the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct).

[0156] To minimize errors and the effect of sample-to-sample variation, RT-PCR is usually performed using an internal standard. The ideal internal standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment. RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and actin, beta (ACTB). For performing analysis on pre-implantation embryos and oocytes, conserved helix -loop-helix ubiquitous kinase (CHUK) is a gene that is used for normalization.

[0157] A more recent variation of the RT-PCR technique is the real time quantitative PCR, which measures PCR product accumulation through a dual-labeled fluorogenic probe (i.e., TAQMAN probe). Real time PCR is compatible both with quantitative competitive PCR, in which internal competitor for each target sequence is used for normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR. For further details see, e.g., Held et al., Genome Research 6:986 994 (1996), the contents of which are incorporated by reference herein in their entirety.

[0158] In another embodiment, a Mass ARRAY -based gene expression profiling method is used to measure gene expression. In the MassARRAY-based gene expression profiling method, developed by Sequenom, Inc. (San Diego, Calif.) following the isolation of RNA and reverse transcription, the obtained cDNA is spiked with a synthetic DNA molecule (competitor), which matches the targeted cDNA region in all positions, except a single base, and serves as an internal standard. The cDNA / competitor mixture is PCR amplified and is subjected to a post -PCR shrimp alkaline phosphatase (SAP) enzyme treatment, which results in the dephosphorylation of the remaining nucleotides. After inactivation of the alkaline phosphatase, the PCR products from the competitor and cDNA are subjected to primer extension, which generates distinct mass signals for the competitor- and cDNA-derives PCR products. After purification, these products are dispensed on a chip array, which is pre-loaded with components needed for analysis with matrix- assisted laser desorption ionization time-of -flight mass spectrometry (MALDI-TOF MS) analysis. The cDNA present in the reaction is then quantified by analyzing the ratios of the peak areas in the mass spectrum generated. For further details see, e.g., Ding and Cantor, Proc. Natl. Acad. Sci. USA 100:3059 3064 (2003). Further PCR-based techniques include, for example, differential display (Liang and Pardee, Science 257:967 971 (1992)); amplified fragment length polymorphism (iAFLP) (Kawamoto et al., Genome Res. 12:1305 1312 (1999)); BeadArrayTM technology (Illumina, San Diego, Calif.; Oliphant et al., Discovery of Markers for Disease (Supplement to Biotechniques), June 2002; Ferguson et al., Analytical Chemistry 72:5618 (2000)); BeadsArray for Detection of Gene Expression (BADGE), using the commercially available LuminexlOO LabMAP system and multiple color-coded microspheres (Luminex Corp., Austin, Tex.) in a rapid assay for gene expression (Yang et al., Genome Res. 11 :1888 1898 (2001)); and high coverage expression profiling (HiCEP) analysis (Fukumura et al., Nucl. Acids. Res. 31(16) e94 (2003)). The contents of each of which are incorporated by reference herein in their entirety.

[0159] In certain embodiments, differential gene expression can also be identified, or confirmed using a microarray technique. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. Methods for making microarrays and determining gene product expression (e.g., RNA or protein) are shown in U.S. Patent Publication No. 2006 / 0195269), the content of which is incorporated by reference herein in its entirety.

[0160] In a specific embodiment of the microarray technique, PCR amplified inserts of cDNA clones are applied to a substrate in a dense array, for example, at least 10,000 nucleotide sequences are applied to the substrate. The microarrayed genes, immobilized on the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pair-wise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels, as described in, for example, Schena et al., Proc. Natl. Acad. Sci. USA 93(2):106 149 (1996), the contents of which are incorporated by reference herein in their entirety. Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Incyte's microarray technology.

[0161] Alternatively, protein levels can be determined by constructing an antibody microarray in which binding sites comprise immobilized, preferably monoclonal, antibodies specific to a plurality of protein species encoded by the cell genome. Preferably, antibodies are present for a substantial fraction of the proteins of interest. Methods for making monoclonal antibodies are well known (see, e.g., Harlow and Lane, 1988, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor, N.Y., which is incorporated in its entirety for all purposes). In one embodiment, monoclonal antibodies are raised against synthetic peptide fragments designed based on genomic sequence of the cell. With such an antibody array, proteins from the cell are contacted to the array, and their binding is assayed with assays known in the art. Generally, the expression, and the level of expression, of proteins of diagnostic or prognostic interest can be detected through immunohistochemical staining of tissue slices or sections.

[0162] Finally, levels of transcripts of marker genes in a number of tissue specimens may be characterized using a “tissue array” as described in, for example, Kononen et al., Nat. Med 4(7):844-7 (1998). In a tissue array, multiple tissue samples are assessed on the same microarray. The arrays allow in situ detection of RNA and protein levels; consecutive sections allow the analysis of multiple samples simultaneously.

[0163] In other embodiments, Serial Analysis of Gene Expression (SAGE) is used to measure gene expression. Serial analysis of gene expression (SAGE) is a method that allows the simultaneous and quantitative analysis of a large number of gene transcripts, without the need of providing an individual hybridization probe for each transcript. First, a short sequence tag (about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript. Then, many transcripts are linked together to form long serial molecules, that can be sequenced, revealing the identity of the multiple tags simultaneously. The expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags, and identifying the gene corresponding to each tag. For more details see, e.g., Velculescu et al., Science 270:484 487 (1995); and Velculescu et al., Cell 88:243 51 (1997), the contents of each of which are incorporated by reference herein in their entirety.

[0164] In other embodiments Massively Parallel Signature Sequencing (MPSS) is used to measure gene expression. This method, described by Brenner et al., Nature Biotechnology 18:630 634 (2000), is a sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 pm diameter microbeads. First, a microbead library of DNA templates is constructed by in vitro cloning. This is followed by the assembly of a planar array of the template-containing microbeads in a flow cell at a high density (typically greater than 3 x 106microbeads / cm2). The free ends of the cloned templates on each microbead are analyzed simultaneously, using a fluorescence-based signature sequencing method that does not require DNA fragment separation. This method has been shown to simultaneously and accurately provide, in a single operation, hundreds of thousands of gene signature sequences from a yeast cDNA library.

[0165] Immunohistochemistry methods are also suitable for detecting the expression levels of the gene products of the present invention. Thus, antibodies (monoclonal or polyclonal) or antisera, such as polyclonal antisera, specific for each marker are used to detect expression. The antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibody is used in conjunction with a labeled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.

[0166] In certain embodiments, a proteomics approach is used to measure gene expression. A proteome refers to the totality of the proteins present in a sample (e.g., tissue, organism, or cell culture) at a certain point of time. Proteomics includes, among other things, study of the global changes of protein expression in a sample (also referred to as expression proteomics). Proteomics typically includes the following steps: (1) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) identification of the individual proteins recovered from the gel, e.g., my mass spectrometry or N-terminal sequencing, and (3) analysis of the data using bioinformatics. Proteomics methods are valuable supplements to other methods of gene expression profiling, and can be used, alone or in combination with other methods, to detect the products of the diagnostic markers of the present invention.

[0167] In some embodiments, mass spectrometry (MS) analysis can be used alone or in combination with other methods (e g., immunoassays or RNA measuring assays) to determine the presence and / or quantity of the one or more biomarkers disclosed herein in a biological sample. In some embodiments, the MS analysis includes matrix -assisted laser desorption / ionization (MALDI) time-of-flight (TOF) MS analysis, such as for example direct- spot MALDI-TOF or liquid chromatography MALDI-TOF mass spectrometry analysis. In some embodiments, the MS analysis comprises electrospray ionization (ESI) MS, such as for example liquid chromatography (LC) ESI-MS. Mass analysis can be accomplished using commercially- available spectrometers. Methods for utilizing MS analysis, including MALDI-TOF MS and ESI-MS, to detect the presence and quantity of biomarker peptides in biological samples are known in the art. See, for example, U.S. Patent Nos. 6,925,389; 6,989,100; and 6,890,763, each of which is incorporated by reference herein in their entirety.

[0168] Reports on genetic modifiers of LRRK2

[0169] Methods of the invention may include providing a report on the subject. The report may identify one or more genetic modifiers of LRRK2 in the genetic data from the subject. The report may contain additional information about the subject, such as age, sex, weight, height, genetic data, genomic data, or other health or medical information. The report may include other information related to PD. For example and without limitation, the report may contain information about symptoms of PD or genes associated with PD, such as the symptoms and genes described above.

[0170] The report may be provided in any suitable manner. For example and without limitation, the report may be provided on paper or on a display device, such as a computer monitor, telephone, portable electronic device, or the like.

[0171] The report may be provided to a healthcare provider, such as a physician or nurse. The report may provide the healthcare provider guidance on whether treatment of the subject with a LRRK2 inhibitor is appropriate. The report may provide the healthcare provider with instructions or recommendations for treating the subject with a LRRK2 inhibitor. The report may recommend that the healthcare provider prescribe or provide a LRRK2 inhibitor for the subject or otherwise instruct the subject to procure and take a LRRK2 inhibitor.

[0172] The report may include guidance on whether to use a second agent in addition to a LRRK2 inhibitor to treat the subject. The second agent may be a known therapeutic agent for treatment of PD, such as any of those described above.

[0173] LRRK2 inhibitors

[0174] Methods of the invention may include providing one or more LRRK2 inhibitors to a subject or recommending that a subject take one or more LRRK2 inhibitors. LRRK2 inhibitors are known in the art and described in, for example, International Patent Publication Nos. WO 2012 / 028629, WO 2012 / 058193, WO 2012 / 118679, WO 2012 / 143143, WO 2012 / 143144, WO

[0175] 2014 / 001973, WO 2014 / 060112, WO 2014 / 060113, WO 2014 / 145909, WO 2014 / 160430, WO

[0176] 2014 / 170248, WO 2015 / 092592, WO 2015 / 113451, WO 2015 / 113452, WO 2016 / 130920, WO

[0177] 2017 / 012576, WO 2017 / 046675, WO 2017 / 087905, WO 2017 / 106771, WO 2017 / 156493, WO

[0178] 2017 / 218843, WO 2018 / 137573, WO 2018 / 137593, WO 2018 / 137618, WO 2018 / 137619, WO

[0179] 2018 / 163030, WO 2018 / 163066, WO 2018 / 217946, WO 2019 / 012093, WO 2019 / 104086, WO

[0180] 2019 / 112269, WO 2019 / 126383, WO 2020 / 149723, WO 2020 / 170205, and WO 2020 / 210684;

[0181] U.S. Patent No. 9,499,535; co-pending U.S. Application Nos. 63 / 050,385, 63 / 133,523, 63 / 113,533, 63 / 137,814, 63 / 137816, and 63 / 142009; and co-pending International Application Nos. PCT / IB2020 / 000727, PCT / IB2020 / 000730, PCT / US2021 / 041270, PCT / US2021 / 041271, PCT / US2023 / 080275, and PCT / US2023 / 080295 the contents of each of which are incorporated herein by reference in their entirety. Any LRRK2 disclosed in any of the aforementioned references may be used in methods of the invention.

[0182] For example and without limitation, the LRRK2 inhibitor may be CZC-25146, CZC- 54252, DNL151, DNL201, GNE-7915, GSK2578215A, HG-10-102-01, JH-II-127, K252A, K252B, LRRK2-IN-1, MLi-2, PF-06447475, or staurosporine.

[0183] In certain embodiments, the one or more LRRK2 inhibitors for therapy is selected from a group consisting of: CZC-25146, CZC-54252, DNL151, DNL201, GNE-7915, GSK2578215A, HG-10-102-01, JH-II-127, K252A, K252B, LRRK2-IN-1, MLi-2, PF-06447475, and staurosporine. In certain embodiments, the LRRK2 inhibitors are selected from a group consisting of: formulas (I), (II), (HI), and (IV): wherein:

[0184] A is NH, O, S, C=O, NR3or CR4R5;

[0185] X is an optionally substituted arylene, heteroarylene, cycloalkylene, heterocycloalkylene, alkylcycloalkylene, heteroalkylcycloalkylene, aralkylene or heteroaralkylene group;

[0186] R1is an optionally substituted alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0187] R2is a hydrogen atom, a halogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0188] R3is an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkyl- cycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0189] R4is a hydrogen atom, NO2, Ns, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group; and

[0190] R5is a hydrogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0191] B is NH, O, S, C=O, NR14or CR15R16; R11is an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0192] R12is an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group, wherein R12is bound to the pyrimidine ring of formula (II) via a carbon -carbon bond;

[0193] R13is a hydrogen atom, a halogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0194] R14is an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkyl- cycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0195] R15is a hydrogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0196] R16is a hydrogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;

[0197] R21is aryl or heteroaryl, each of which is optionally substituted;

[0198] R22is H, halo, OH, CN, CF3, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, C1-6 thioalkyl, C3-8 cycloalkyl, C2-8 heterocycloalkyl, aryl, or heteroaryl; and

[0199] Y is aryl or 5- or 6-membered heteroaryl; wherein each of the Ci-6 alkyl, Ci-6 alkoxy, Ci- 6 haloalkyl, C1-6 thioalkyl, C3-8 cycloalkyl, C2-8 heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one or more moieties selected from the group consisting of halo, OH, CN, CF3, NH2, NO2, C1-6 alkyl, C1-6 haloalkyl, C1-6 thioalkyl, C3-8 cycloalkyl, C2-8 heterocycloalkyl, C2-8 heterocycloalkenyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy, C1-6 haloalkoxy, C1-6 alkylamino, C2-6 dialkylamino, C7-12 aralkyl, C1-12 heteroaralkyl, aryl, heteroaryl, -C(O)R, -C(O)OR, -C(O)NRR’, -C(O)NRS(O)2R’, -C(O)NRS(O)2NR’R”, -OR, - OC(O)NRR’, -NRR’, -NRC(O)R’, -NRC(O)NR’R”, -NRS(O)2R’, -NRS(O)2NR’R”, -S(O)2R, and -S(O)2NRR’, in which each of R, R’, and R”, independently, is H, halo, OH, C1-6 alkyl, C1-6 haloalkyl, C 1-6 alkoxy, C3-8 cycloalkyl, C2-8 heterocycloalkyl, aryl, or heteroaryl, or R and R’, orR’ and R”, together with the nitrogen to which they are attached, form C2-8 heterocycloalkyl; R31is C(O)CH2R33, optionally substituted cycloalkyl, optionally substituted cycloheteroalkyl, optionally substituted cycloalkenyl, optionally substituted cycloheteroalkenyl, optionally substituted aryl, or optionally substituted heteroaryl; each instance of R32is independently halo, haloalkyl, optionally substituted alkoxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl;

[0200] R33is optionally substituted cycloalkyl, optionally substituted cycloheteroalkyl, optionally substituted cycloalkenyl, optionally substituted cycloheteroalkenyl, optionally substituted aryl, or optionally substituted heteroaryl;

[0201] Z is cycloalkyl, cycloheteroalkyl, cycloalkenyl, cycloheteroalkenyl, aryl, or heteroaryl; Z may be an aryl substituted with 2 or 3 instances of R2. Z may be a phenyl substituted with 2 or 3 instances of R2. Z may be a heteroaryl substituted with 2 or 3 instances of R2. Z may be a sixmembered heteroaryl substituted with 2 or 3 instances of R2; and n is 0-5, or a pharmaceutically acceptable salt of any compound described above.

[0202] In one aspect, the invention provides a compound of Formula (V): or an enantiomer, mixture of enantiomers, tautomer, or pharmaceutically acceptable salt thereof, wherein: n is 1, 2, or 3; Y i and Y2 are independently N or C;

[0203] Zi, Z2, and Z3 are independently selected from H, -OH, halo, cyano, amino, Ci-Ce alkyl, C1-C6 heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxy alkoxy;

[0204] X is H, halo, cyano, Ci-Ce alkyl, optionally deuterated C1-C6 alkyl, C3-C6 cycloalkyl, Ci- Ce heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxy alkoxy;

[0205] Ri, R2, and R4 are independently selected from H, halo, cyano, Ci-Ce alkyl, optionally deuterated Ci-Ce alkyl, Ci-Ce heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxyalkoxy, with the proviso that these substitutions are permitted by valency;

[0206] W is H or C1-C4 substituted or unsubstituted alkyl, wherein W may optionally form a ring with Y2 when Y2 is C;

[0207] L is a linker, wherein L is a single bond, substituted or unsubstituted C 1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, wherein the one or more heteroatoms are selected from O, S, or N;

[0208] A is 4-8 membered substituted or unsubstituted heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, wherein one or more heteroatoms are selected from a group consisting of O, S, orN; and wherein the substituents may be selected from a group consisting of substituted or unsubstituted 3-7 membered heterocycle, C1-C4 alkyl, -CH2-cycloalkyl, -CF2-cycloalky, -C(=O)- O-alkyl, halo, deuterium, cyano, cyanoalkyl, -CF3, mono-, di-, or tri-halo alkyl, CH(CH3)- cycloalkyl, -CH2-aryl, -CF2-aryl, -CH(-CH3)-aryl, C(=O)-alkyl, -C(=O)cycloalkyl, -C(=O)-NH- alkyl, -COOH (and esters and carboxamides thereof), -C(=O)-morpholine, -C(=O)-heterocycles, -C(-CH3)2-OH, -CH2-C(=O)-NH2; -hydroxy, alkylhydroxy, alkyl-COOH (and esters and carboxamides thereof), amino, -NHC(=O)alkyl, -N(alkyl)C(=O)alkyl, -NHC(=O)aryl, - N(alkyl)C(=O)aryl, substituted or unsubstituted morpholine, 3-7 membered heterocycle, any of which may have one or more substituents, 3-7 membered cycloalkyl or heterocycle, wherein the 3-7 membered cycloalkyl or heterocycle is optionally fused with another 3-7 cycloalkyl or heterocycle, wherein the rings are spiro, bridged bicyclic, or spiro, wherein the at least one heteroatom in the heterocycle rings are independently selected from O, S, and N; and wherein one or more hydrogen atoms are optionally deuterium.

[0209] In another embodiment, in the compound of Formula (V), Yi is N and Y2 is C.

[0210] In another embodiment, in the compound of Formula (V), Yi is C and Y2 is N.

[0211] In another embodiment, in the compound of Formula (V), Yi is C and Y2 is C.

[0212] In another embodiment, in the compound of Formula (V), X is selected from a group consisting of -CH3, -CH2-CH3, -CD3, H, and F.

[0213] In another embodiment, in the compound of Formula (V), Zi, Z2, and Z3are independently selected from H, F, or Cl.

[0214] In another embodiment, in the compound of Formula (V), Zi is F and Z2 is F.

[0215] In another embodiment, Z in the compound of Formula (V),3is H.

[0216] In another embodiment, in the compound of Formula (V), Z3is F.

[0217] In another embodiment, in the compound of Formula (V), Ri is selected from H, F, -CH3, -CH2-CH3, -CF3, or -CHF2.

[0218] In another embodiment, in the compound of Formula (V), R2 is selected from a group consisting of H or F.

[0219] In another embodiment, in the compound of Formula (V), R2 is F.

[0220] In another embodiment, in the compound of Formula (V), R4 is H.

[0221] In another embodiment, in the compound of Formula (V), L is a single bond.

[0222] In another embodiment, in the compound of Formula (V), L is alkyl.

[0223] In another embodiment, in the compound of Formula (V), L is -C=C-.

[0224] In another embodiment, in the compound of Formula (V), L is -NH-CH2-.

[0225] In another embodiment, in the compound of Formula (V), A is not substituted or unsubstituted 1,2,3,6-Tetrahydropyridin.

[0226] In another embodiment, in the compound of Formula (V), A is selected from:

[0227]

[0228]

[0229] In another embodiment, the compound of Formula (V) is selected from:

[0230]

[0231] In one aspect, the invention provides a compound of Formula (VI): or an enantiomer, mixture of enantiomers, tautomer, or pharmaceutically acceptable salt thereof, wherein: n is 1, 2, or 3;

[0232] Y is N or CR2;

[0233] Zi, Z2, and Z3 are independently selected from H, -OH, halo, cyano, amino, Ci-Ce alkyl, C1-C6 heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxy alkoxy;

[0234] X is H, halo, cyano, cycloalkyl, Ci-Ce alkyl, optionally deuterated Ci-Ce alkyl, Ci-Ce heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxyalkoxy;

[0235] Ri, R2, and R4 are independently selected from H, halo, cyano, Ci-Ce alkyl, optionally deuterated Ci-Ce alkyl, Ci-Ce heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxy alkoxy;

[0236] L is a linker, wherein L is a single bond, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, wherein the one or more heteroatoms are selected from O, S, or N;

[0237] A is 4-8 membered substituted or unsubstituted heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, wherein one or more heteroatoms are selected from a group consisting of O, S, or N; and wherein the substituents may be selected from a group consisting of substituted or unsubstituted 3-7 membered heterocycle, =0, -CH2-cycloalkyl, -CF2-cycloalky, -C(=O)-O-alkyl, halo, deuterium, cyano, cyanoalkyl, -CF3, mono-, di-, or tri-halo alkyl, CH(CH3)-cycloalkyl, - CH2-aiyl, -CF2-aryl, -CH(-CH3)-aryl, C(=O)-alkyl, -C(=O)cycloalkyl, -C(=O)-NH-alkyl, - COOH (and esters and carboxamides thereof), -C(=O)-morpholine, -C(=O)-heterocycles, -C(- CH3)2-OH, -CH2-C(=O)-NH2; -hydroxy, alkylhydroxy, alkyl-COOH (and esters and carboxamides thereof), amino, NHC(=O)alkyl, -N(alkyl)C(=O)alkyl, -NHC(=O)aryl, - N(alkyl)C(=O)aryl, -N(H)S(=O)2alkyl, -N(H)S(=O)2aryl, -N(alkyl)S(=O)2alkyl, cyanoalkyl, haloalkyl, -S=(0)2-alkyl, -S=(O)2-cycloalkyl, -S=(0)2-aryl, -S(=0)2N(H)alkyl, substituted or unsubstituted morpholine, 3-7 membered heterocycle, any of which may have one or more substituents, 3-7 membered cycloalkyl or heterocycle, wherein the 3-7 membered cycloalkyl or heterocycle is optionally fused with another 3-7 cycloalkyl or heterocycle, wherein the rings are spiro, bridged bicyclic, or spiro, wherein the at least one heteroatom in the heterocycle rings are independently selected from O, S, and N; and wherein one or more hydrogen atoms are optionally deuterium.

[0238] In another embodiment, in compounds of Formula (VI), Y is N.

[0239] In another embodiment, in compounds of Formula (VI), Y is CR2.

[0240] In another embodiment, in compounds of Formula (VI), Zi and Z2 are F.

[0241] In another embodiment, in compounds of Formula (VI), Zi and Z2 are independently selected from Cl or F.

[0242] In another embodiment, in compounds of Formula (VI), Z3 is H.

[0243] In another embodiment, in compounds of Formula (VI), Z3 is F.

[0244] In another embodiment, in compounds of Formula (VI), Z3 is -CH2-OH.

[0245] In another embodiment, in compounds of Formula (VI), X is selected from a group consisting of methyl, H, Cl, or -CD3.

[0246] In another embodiment, in compounds of Formula (VI), X is methyl.

[0247] In another embodiment, in compounds of Formula (VI), Ri is selected from H or methyl.

[0248] In another embodiment, in compounds of Formula (VI), R2 is H.

[0249] In another embodiment, in compounds of Formula (VI), R4 is H.

[0250] In another embodiment, in compounds of Formula (VI), L is a single bond.

[0251] In another embodiment, in compounds of Formula (VI), L is alkyl.

[0252] In another embodiment, in compounds of Formula (VI), L is -C=C-

[0253] In another embodiment, in compounds of Formula (VI), A is not substituted or unsubstituted 1,2,3,6-Tetrahydropyridin.

[0254] In another embodiment, in compounds of Formula (VI), A is morpholine with optional substitutions

[0255] In another embodiment, in compounds of Formula (VI), A is selected from: The LRRK2 inhibitor may be provided to a subject in a pharmaceutical composition. The pharmaceutical composition may contain the LRRK2 inhibitor in a therapeutically effective amount. A therapeutically effective amount means an amount that is effective to prevent, alleviate, or ameliorate symptoms of a disease, such as PD, or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the skill in the art. The therapeutically effective amount or dosage of a LRRK2 inhibitor can vary within wide limits and may be determined in a manner known in the art. Such dosage may be adjusted to the individual requirements in each particular case including the specific compound being administered, the route of administration, the condition being treated, as well as the patient being treated.

[0256] For oral administration such therapeutically useful agents can be administered by one of the following routes: oral, e.g., as tablets, dragees, coated tablets, pills, semisolids, soft or hard capsules, for example, soft and hard gelatin capsules, aqueous or oily solutions, emulsions, suspensions or syrups, parenteral including intravenous, intramuscular and subcutaneous injection, e.g., as an injectable solution or suspension, rectal as suppositories, by inhalation or insufflation, e.g., as a powder formulation, as microcrystals or as a spray (e.g., liquid aerosol), transdermal, for example via an transdermal delivery system (TDS) such as a plaster containing the active ingredient or intranasal. For the production of such tablets, pills, semisolids, coated tablets, dragees and hard, e.g., gelatin, capsules, the therapeutically useful product may be mixed with pharmaceutically inert, inorganic or organic excipients as are e.g., lactose, sucrose, glucose, gelatin, malt, silica gel, starch or derivatives thereof, talc, stearinic acid or their salts, dried skim milk, and the like. For the production of soft capsules one may use excipients as are e.g., vegetable, petroleum, animal or synthetic oils, wax, fat, polyols. For the production of liquid solutions, emulsions or suspensions or syrups one may use as excipients e.g., water, alcohols, aqueous saline, aqueous dextrose, polyols, glycerin, lipids, phospholipids, cyclodextrins, vegetable, petroleum, animal or synthetic oils. Particularly useful are lipids, such as phospholipids (e.g., natural origin and / or with a particle size between 300 to 350 nm) in phosphate buffered saline (pH = 7 to 8, e.g., 7.4). For suppositories one may use excipients as are e.g., vegetable, petroleum, animal or synthetic oils, wax, fat and polyols. For aerosol formulations one may use compressed gases suitable for this purpose, as are e.g., oxygen, nitrogen and carbon dioxide. The pharmaceutically useful agents may also contain additives for conservation, stabilization, e.g., UV stabilizers, emulsifiers, sweetener, aromatizers, salts to change the osmotic pressure, buffers, coating additives and antioxidants.

[0257] Providing a LRRK2 inhibitor to a subject

[0258] Methods of the invention may include providing a LRRK2 inhibitor to a subject. The LRRK2 inhibitor may be provided by any suitable route or mode of administration. For example and without limitation, the compound may be provided buccally, dermally, enterally, intraarterially, intramuscularly, intraocularly, intravenously, nasally, orally, parenterally, pulmonarily, rectally, subcutaneously, topically, transdermally, by injection, or with or on an implantable medical device (e.g., stent or drug-eluting stent or balloon equivalents).

[0259] The LRRK2 inhibitor may be provided according to a dosing regimen. A dosing regimen may include a dosage, a dosing frequency, or both.

[0260] Doses may be provided at any suitable interval. For example and without limitation, doses may be provided once per day, twice per day, three times per day, four times per day, five times per day, six times per day, eight times per day, once every 48 hours, once every 36 hours, once every 24 hours, once every 12 hours, once every 8 hours, once every 6 hours, once every 4 hours, once every 3 hours, once every two days, once every three days, once every four days, once every five days, once every week, twice per week, three times per week, four times per week, or five times per week.

[0261] The dose may be provided in a single dosage, i.e., the dose may be provided as a single tablet, capsule, pill, etc. Alternatively, the dose may be provided in a divided dosage, i.e., the dose may be provided as multiple tablets, capsules, pills, etc.

[0262] The dosing may continue for a defined period. For example and without limitation, doses may be provided for at least one week, at least two weeks, at least three weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, at least twelve weeks or more.

[0263] The subject may be any type of subject, such as any of those described above in relation to assays to obtain genetic data.

[0264] The invention includes combination therapies in which a LRRK2 inhibitor is provided to a subject in combination with a second agent, such as any of the drugs described above in the section on PD. The LRRK2 inhibitor and the second agent may be provided in a single composition, or they may be provided in separate compositions. The LRRK2 inhibitor and the second agent may be provided according to the same dosing regimen, or they may be provided according to different dosing regimens.

[0265] Examples

[0266] Example 1

[0267] Likelihood of responsiveness to LRRK2 inhibitors was analyzed in a population of human subjects. GlcCer / Cer ratios were measured in the CSF of patients diagnosed as having PD. The GlcCer / Cer ratio was calculated by using the sum of the following glucosylceramide species divided by the sum of the following ceramide species GlcCer (dl8:l, 16:0), GlcCer (d l8:l, 18:0), GlcCer (dl8:l, 24:0), GlcCer (d l8:l, 24:1) and Cer(d 18: 1 / 16:0), Cer(d 18:1 / 18:0), Cer(d 18: 1 / 24:0), Cer(dl8:l / 24:1).

[0268] FIG. 1 provides the ratio of GlcCer to Cer in different sets of patient populations.

[0269] Results are summarized below:

[0270] Summary:

[0271] Mean Comparison - general linear model (GLM) controlled for Age and Sex

[0272] An elevated GlcCer / Cer ratio, for example greater than 2:1 was predictive of LRRK2 associated Parkinson’s disease. Further elevated GlcCer / Cer ratios, for example greater than 2.15 was predictive of Parkinson’s disease associated with LRRK2 rare variants.

[0273] FIG. 2A-C provides ratios of GlcCer to Cer in different sets of patient populations.

[0274] GlcCer to Cer ratios stratified upwards in subject with LRRK2 rare variants and Parkinson’s Disease patients but not in health controls (HC) with or without a LRRK2 rare variant. GlcCer to Cer ratios also stratified upwards in subjects predicted to have Parkinson’s and a LRRK2 mutation in comparison with subjects without a LRRK2 mutation. The data together shows a relationship between GlcCer to Cer ratios in LRRK2 driven Parkinson’s Disease. Rare variant carriers separate from subjects without rare variants in PD, but not healthy controls.

[0275] Notably, the LRRK2-prediction was stratified in two independent patient populations (LCC 122 and PPMI 135).

[0276] The data validates a diagnostic model for identifying LRRK2 rare variants using GlcCer to Cer rations and LRKK2 Parkinson’s Disease.

[0277] Incorporation by Reference

[0278] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0279] Equivalents

[0280] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification, and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

1. ClaimsWhat is claimed is:

1. A method of treating a patient having Parkinson’s disease associated with leucine rich repeat kinase (LRRK2), the method comprising: providing one or more LRRK2 inhibitors to the patient that presents with Parkinson’s disease and an elevated ratio of glucosylceramide (GlcCer) to ceramide (Cer) compared to the GlcCerCer ratio of a subject not suffering from Parkinson’s disease, thereby treating Parkinson’s disease associated with LRRK2.

2. The method of claim 1, wherein the ceramide species is be selected from the group consisting of (i) Cer(dl8: 1 / 16:0), (ii) Cer (d 18: 1 / 18:0), (iii) Cer (d 18: 1 / 24:0), (iv) Cer (d 18: 1 / 24: 1), or a combination thereof; and the GlcCer species is selected from the group consisting of (i) GlcCer (d 18 : 1 , 16:0), (ii) GlcCer (dl8:l, 18:0), (iii) GlcCer (dl8:l, 24:0), (iv) GlcCer (dl8:l, 24:1), or a combination thereof.

3. The method of claim 1, wherein the GlcCerCer ratio is measured in a biofluid of the patient.

4. The method of claim 3, wherein the biofluid is urine, blood, cerebrospinal fluid (CSF), bile, or saliva.

5. The method of claim 1, wherein the elevated GlcCerCer ratio is at a concentration which indicates that the patient would be responsive to the one or more LRRK2 inhibitors.

6. The method of claim 1, wherein one or more LRRK2 inhibitors are selected from the group consisting of CZC-25146, CZC-54252, DNL151, DNL201, GNE-7915, GSK2578215A, HG-10-102-01, JH-II-127, K252A, K252B, LRRK2-IN-1, MLi-2, PF-06447475, and staurosporine.

7. The method of claim 1, wherein one or more LRRK2 inhibitors are selected from the group consisting of formulas (I), (II), (III), and (IV):wherein:A is NH, O, S, C=O, NR3or CR4R5;X is an optionally substituted arylene, heteroarylene, cycloalkylene, heterocycloalkylene, alkylcycloalkylene, heteroalkylcycloalkylene, aralkylene or heteroaralkylene group;R1is an optionally substituted alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;R2is a hydrogen atom, a halogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;R3is an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkyl- cycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;R4is a hydrogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group; andR5is a hydrogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;B is NH, O, S, C=O, NR14or CR15R16;R11is an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;R12is an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group, wherein R12is bound to the pyrimidine ring of formula (II) via a carbon-carbon bond;R13is a hydrogen atom, a halogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;R14is an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkyl- cycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;R15is a hydrogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;R16is a hydrogen atom, NO2, N3, OH, SH, NH2 or an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group;R21is aryl or heteroaryl, each of which is optionally substituted;R22is H, halo, OH, CN, CF3, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, C1-6 thioalkyl, C3-8 cycloalkyl, C2-8 heterocycloalkyl, aryl, or heteroaryl; andY is aryl or 5- or 6-membered heteroaryl; wherein each of the C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, C1-6 thioalkyl, C3-8 cycloalkyl, C2-8 heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one or more moieties selected from the group consisting of halo, OH, CN, CF3, NH2, NO2, C1-6 alkyl, C1-6 haloalkyl, C1-6 thioalkyl, C3 8 cycloalkyl, C2-8 heterocycloalkyl, C2-8 heterocycloalkenyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy, C1-6 haloalkoxy, C1-6 alkylamino, C2-6 dialkylamino, C7-12 aralkyl, C1-12 heteroaralkyl, aryl, heteroaryl, -C(O)R, -C(O)OR, -C(O)NRR’, -C(O)NRS(O)2R’, -C(O)NRS(O)2NR’R”, -OR, -OC(O)NRR’, -NRR’, -NRC(O)R’, -NRC(O)NR’R”, - NRS(O)2R’, -NRS(O)2NR’R”, -S(O)2R, and -S(O)2NRR’, in which each of R, R’, and R”, independently, is H, halo, OH, Ci-6 alkyl, Ci-6 haloalkyl, Ci-6 alkoxy, C3-8 cycloalkyl, C2-8 heterocycloalkyl, aryl, or heteroaryl, or R and R’, or R’ and R”, together with the nitrogen to which they are attached, form C2-8 heterocycloalkyl;R31is C(O)CH2R33, optionally substituted cycloalkyl, optionally substituted cycloheteroalkyl, optionally substituted cycloalkenyl, optionally substituted cycloheteroalkenyl, optionally substituted aryl, or optionally substituted heteroaryl; each instance of R32is independently halo, haloalkyl, optionally substituted alkoxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl;R33is optionally substituted cycloalkyl, optionally substituted cycloheteroalkyl, optionally substituted cycloalkenyl, optionally substituted cycloheteroalkenyl, optionally substituted aryl, or optionally substituted heteroaryl;Z is cycloalkyl, cycloheteroalkyl, cycloalkenyl, cycloheteroalkenyl, aryl, or heteroaryl; Z may be an aryl substituted with 2 or 3 instances of R2. Z may be a phenyl substituted with 2 or 3 instances of R2. Z may be a heteroaryl substituted with 2 or 3 instances of R2. Z may be a six -membered heteroaryl substituted with 2 or 3 instances of R2; and n is 0-5, or a pharmaceutically acceptable salt of any compound described above.

8. The method of claim 1, wherein one or more LRRK2 inhibitors are selected from the group consisting of formula V:or an enantiomer, mixture of enantiomers, tautomer, or pharmaceutically acceptable salt thereof, wherein: n is 1, 2, or 3;Yi and Y2 are independently N or C;Zi, Z2, and Z3 are independently selected from H, -OH, halo, cyano, amino, Ci-Ce alkyl, C1-C6 heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxy alkoxy;X is H, halo, cyano, Ci-Ce alkyl, optionally deuterated Ci-Ce alkyl, C3-C6 cycloalkyl, Ci- Ce heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxy alkoxy;Ri, R2, and R4 are independently selected from H, halo, cyano, Ci-Ce alkyl, optionally deuterated Ci-Ce alkyl, Ci-Ce heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxyalkoxy, with the proviso that these substitutions are permitted by valency;W is H or C1-C4 substituted or unsubstituted alkyl, wherein W may optionally form a ring with Y2 when Y2 is C;L is a linker, wherein L is a single bond, substituted or unsubstituted C 1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, wherein the one or more heteroatoms are selected from O, S, or N;A is 4-8 membered substituted or unsubstituted heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, wherein one or more heteroatoms are selected from a group consisting of O, S, orN; and wherein the substituents may be selected from a group consisting of substituted or unsubstituted 3-7 membered heterocycle, C1-C4 alkyl, -CH2-cycloalkyl, -CF2-cycloalky, -C(=O)- O-alkyl, halo, deuterium, cyano, cyanoalkyl, -CF3, mono-, di-, or tri-halo alkyl, CH(CH3)- cycloalkyl, -CH2-aryl, -CF2-aryl, -CH(-CH3)-aiyl, C(=O)-alkyl, -C(=O)cycloalkyl, -C(=O)-NH- alkyl, -COOH (and esters and carboxamides thereof), -C(=O)-morpholine, -C(=O)-heterocycles, -C(-CH3)2-OH, -CH2-C(=O)-NH2; -hydroxy, alkylhydroxy, alkyl-COOH (and esters and carboxamides thereof), amino, -NHC(=O)alkyl, -N(alkyl)C(=O)alkyl, -NHC(=O)aryl, - N(alkyl)C(=O)aryl, substituted or unsubstituted morpholine, 3-7 membered heterocycle, any of which may have one or more substituents, 3-7 membered cycloalkyl or heterocycle, wherein the 3-7 membered cycloalkyl or heterocycle is optionally fused with another 3-7 cycloalkyl or heterocycle, wherein the rings are spiro, bridged bicyclic, or spiro, wherein the at least one heteroatom in the heterocycle rings are independently selected from O, S, and N; and wherein one or more hydrogen atoms are optionally deuterium.

9. A method of claim 1, wherein one or more LRRK2 inhibitors are selected from the group consisting of formula VI:or an enantiomer, mixture of enantiomers, tautomer, or pharmaceutically acceptable salt thereof, wherein: n is 1, 2, or 3;Y is N or CR2;Zi, Z2, and Z3 are independently selected from H, -OH, halo, cyano, amino, Ci-Ce alkyl, C1-C6 heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxy alkoxy;X is H, halo, cyano, cycloalkyl, Ci-Ce alkyl, optionally deuterated Ci-Ce alkyl, Ci-Ce heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxyalkoxy;Ri, R2, and R4 are independently selected from H, halo, cyano, Ci-Ce alkyl, optionally deuterated Ci-Ce alkyl, Ci-Ce heteroalkyl, haloalkyl, alkoxy, haloalkxoxy, -CH(OH)-alkyl, hydroxyalkyl, or hydroxy alkoxy;L is a linker, wherein L is a single bond, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, wherein the one or more heteroatoms are selected from O, S, or N;A is 4-8 membered substituted or unsubstituted heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, wherein one or more heteroatoms are selected from a group consisting of O, S, or N; and wherein the substituents may be selected from a group consisting of substituted or unsubstituted 3-7 membered heterocycle, =0, -CH2-cycloalkyl, -CF2-cycloalky, -C(=O)-O-alkyl, halo, deuterium, cyano, cyanoalkyl, -CF3, mono-, di-, or tri-halo alkyl, CH(CH3)-cycloalkyl, - CH2-aiyl, -CF2-aryl, -CH(-CH3)-aryl, C(=O)-alkyl, -C(=O)cycloalkyl, -C(=O)-NH-alkyl, - COOH (and esters and carboxamides thereof), -C(=O)-morpholine, -C(=O)-heterocycles, -C(- CH3)2-OH, -CH2-C(=O)-NH2; -hydroxy, alkylhydroxy, alkyl-COOH (and esters and carboxamides thereof), amino, NHC(=O)alkyl, -N(alkyl)C(=O)alkyl, -NHC(=O)aryl, - N(alkyl)C(=O)aryl, -N(H)S(=O)2alkyl, -N(H)S(=O)2aryl, -N(alkyl)S(=O)2alkyl, cyanoalkyl, haloalkyl, -S=(O)2-alkyl, -S=(O)2-cycloalkyl, -S=(0)2-aryl, -S(=O)2N(H)alkyl, substituted or unsubstituted morpholine, 3-7 membered heterocycle, any of which may have one or more substituents, 3-7 membered cycloalkyl or heterocycle, wherein the 3-7 membered cycloalkyl or heterocycle is optionally fused with another 3-7 cycloalkyl or heterocycle, wherein the rings arespiro, bridged bicyclic, or spiro, wherein the at least one heteroatom in the heterocycle rings are independently selected from O, S, and N; and wherein one or more hydrogen atoms are optionally deuterium.

10. A method of determining whether a patient having Parkinson’s disease associated with LRRK2 will respond to a LRRK2 inhibitor, the method comprising: conducting an assay to measure the ratio of GlcCerCer from the patient; generating a report that identifies the GlcCerCer ratio from the patient as compared to the GlcCerCer of a subject not suffering from Parkinson’s disease and having LRRK2; providing the report to a physician such that if the report indicates an elevated GlcCerCer ratio in the patient as compared to the subject, the physician prescribes or provides the patient with one or more LRRK2 inhibitors.

11. The method of claim 10, wherein, the ceramide species is be selected from the group consisting of (i) Cer(dl8:l / 16:0), (ii) Cer (dl8:l / 18:0), (iii) Cer (dl8:l / 24:0), (iv) Cer (d 18:1 / 24:1), or a combination thereof; and the GlcCer species is selected from the group consisting of (i) GlcCer (d 18 : 1 , 16:0), (ii) GlcCer (d 18:1, 18:0), (iii) GlcCer (dl8: 1, 24:0), (iv) GlcCer (d 18:1, 24:1), or a combination thereof.

12. The method of claim 10, wherein the GlcCerCer ratio is measured in a biofluid of the patient.

13. The method of claim 12, wherein the biofluid is urine, blood, cerebrospinal fluid (CSF), bile, or saliva.

14. The method of claim 10, wherein an elevated GlcCerCer ratio in the patient indicates that the patient would be responsive to one or more LRRK2 inhibitors.

15. The method of claim 10, wherein one or more LRRK2 inhibitors are selected from the group consisting of CZC-25146, CZC-54252, DNL151, DNL201, GNE-7915, GSK2578215A, HG-10-102-01, JH-II-127, K252A, K252B, LRRK2-IN-1, MLi-2, PF- 06447475, and staurosporine.

16. The method of claim 10, wherein one or more LRRK2 inhibitors are selected from the group consisting of formulas (I), (II), (III), and (IV).

17. The method of claim 10, wherein one or more LRRK2 inhibitor is a compound of Formula (V).

18. The method of claim 10, wherein one or more LRRK2 inhibitor is a compound of Formula (VI).

19. A method of treating a patient having Parkinson’s disease associated with LRRK2, the method comprising: receiving data that identifies the GlcCerCer ratio from the patient; comparing the data with the GlcCerCer ratio from a subject not having the neurological condition and having LRRK2; prescribing or providing the patient with one or more LRRK2 inhibitors if the patient has elevated GlcCerCer ratio as compared to the subject.

20. The method of claim 19, wherein the GlcCerCer ratio is measured in a biofluid of the patient and the subject.

21. The method of claim 20, wherein the biofluid is urine, blood, cerebrospinal fluid (CSF), bile, or saliva.

22. The method of claim 19, wherein, the ceramide species is be selected from the group consisting of (i) Cer (d 18: 1 / 16:0), (ii) Cer (d 18:1 / 18:0), (iii) Cer (d 18:1 / 24:0), (iv) Cer (d!8: 1 / 24:1), or a combination thereof; andthe GlcCer species is selected from the group consisting of (i) GlcCer (d 18: 1, 16:0), (ii) GlcCer (dl8: 1, 18:0), (iii) GlcCer (d 18: 1, 24:0), (iv) GlcCer (d 18: 1, 24:1), or a combination thereof.

23. The method of claim 19, wherein an elevated GlcCerCer ratio in the patient indicates that the patient would be responsive to one or more LRRK2 inhibitors.

24. The method of claim 19, wherein one or more LRRK2 inhibitors are selected from the group consisting of CZC-25146, CZC-54252, DNL151, DNL201, GNE-7915, GSK2578215A, HG-10-102-01, JH-II-127, K252A, K252B, LRRK2-IN-1, MLi-2, PF- 06447475, and staurosporine.

25. The method of claim 19, wherein one or more LRRK2 inhibitors are selected from the group consisting of formulas (I), (II), (III), and (IV).

26. The method of claim 19, wherein one or more LRRK2 inhibitors is a compound of Formula (V) or a pharmaceutically acceptable salt thereof.

27. The method of claim 19, wherein one or more LRRK2 inhibitors is a compound of Formula (VI) or a pharmaceutically acceptable salt thereof.

28. The methods of claims 1, 10, or 19, wherein the one or more LRRK2 compounds selected from the group consisting of :

29. The methods of claims 1, 10, or 19, wherein the one or more LRRK2 compounds selected from the group consisting of :

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