Methods and a kit for detecting parkinson's disease with and without dementia

Abstract

The present invention is directed to methods for diagnosing Parkinson's disease (PD) with or without dementia in a subject. The present invention is further directed to methods for determining the severity of PD in a subject afflicted therewith, for determining responsiveness to anti PD therapy of a subject administered therewith, and for treating a subject afflicted with PD.

Description

METHODS AND A KIT FOR DETECTING PARKINSON’S DISEASE WITH AND WITHOUT DEMENTIAREFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (HUJI-P-0127-PCT.xml; size: 190,416 bytes; and date of creation: 7 November 2024) is herein incorporated by reference in its entirety.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 607,794, titled “METHOD AND KIT FOR DETECTING PARKINSON’S DISEASE WITH AND WITHOUT DEMENTIA”, filed December 8, 2023, the contents of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION

[0003] The present invention relates generally to the field of Parkinson's disease diagnosis.BACKGROUND

[0004] Parkinson's disease (PD) is the second most common neurodegenerative disease, with an expectancy of 14 million patients worldwide by 2025. Although it has been vastly researched over the past century, knowledge of the disease mechanism remains lacking. PD is often described as a movement disability disorder, although recent discoveries have emphasized the non-motor symptoms as well.

[0005] Importantly, PD is twice more prevalent in men than in women and men develop the disease earlier in life. Women and men patients further differ in symptoms, progression, physiopathology, reaction to PD drugs and more. The differences are not fully understood and are attributed to genetic, hormonal and environmental factors. For example, women show more mood impairments while men report more sexual dysfunction. There is anongoing debate as to whether PD male patients suffer more from dementia, but there is an agreement that PD female patients develop it in a later stage.

[0006] Treatment for PD today consists of dopamine substitution with L-Dopa and Carbidopa, with or without subthalamic deep brain stimulation (DBS) that is sometimes given after the "L-Dopa honeymoon" ends. Both treatments aim to alleviate the motor symptoms of the disease, which are believed to be caused by the death of dopaminergic neurons in the Substantia Nigra Pars Compacta. When a person is diagnosed with PD, 50%- 70% of those neurons have already died, therefore the available treatment today can only limit symptoms rather than achieve remission.

[0007] The growing interest in the non-motoric symptoms of PD spans sleep disorders, cognitive impairment, mood disorders, autonomic dysfunction, and pain. There is a growing body of evidence on the possible involvement of the cholinergic system in those symptoms, especially regarding cognitive impairments and autonomic dysfunction. Circulating RNAs have been researched in the past years as an upcoming biomarker in PD in particular. Previous research raised several promising candidates for biomarkers of the disease, however, the use of the rare circular RNAs and small non-coding RNAs such as microRNAs is rather complex, complicating the construction of an easy diagnosis tool for clinical use.

[0008] There is still a great need for early diagnosis based on reliable biomarkers, which may help interfere with specific aspects of the disease course as early as possible.SUMMARY

[0009] According to the first aspect, there is provided a method for diagnosing Parkinson’s disease (PD) in a subject, the method comprising determining in a sample obtained or derived from the subject a level of at least one RNA polynucleotide sequence listed under any one of Tables 1-5, wherein a modulated level of the at least one RNA polynucleotide sequence compared to a control, is indicative of the subject being afflicted with PD, thereby diagnosing PD in the subject.

[0010] According to another aspect, there is provided a method for diagnosing PD with dementia in a subject, the method comprising determining in a sample obtained or derived from the subject a level of at least one RNA polynucleotide sequence listed under any oneof Tables 1 and 2, wherein a modulated level of the at least one RNA polynucleotide sequence compared to a control, is indicative of the subject being afflicted with PD with dementia, thereby diagnosing PD with dementia in the subject.[Oi l] According to another aspect, there is provided a method for diagnosing PD without dementia in a subject, the method comprising determining in a sample obtained or derived from the subject a level of at least one RNA polynucleotide sequence listed under Table 3, wherein a reduced level of the at least one RNA polynucleotide sequence compared to a control, is indicative of the subject being afflicted with PD without dementia, thereby diagnosing PD without dementia in the subject.

[0012] According to another aspect, there is provided a method for determining the severity of PD in a subject afflicted therewith, the method comprising: (a) determining in a sample obtained or derived from the subject a level of at least one RNA polynucleotide sequence listed under any one of Tables 1-4; and (b) comparing the determined level of the at least one RNA polynucleotide sequence to a pre-determined threshold, wherein any one of: (1) the determined level of the at least one RNA polynucleotide sequence listed under any one of Tables 1, 3, and 4 being lower than the pre-determined threshold or the determined level of the at least one RNA polynucleotide sequence listed under Table 2 being greater than the pre-determined threshold is indicative of the subject being afflicted with an immoderate form of PD; (2) the determined level of the at least one RNA polynucleotide sequence listed under any one of Tables 1-4 being equal to the pre-determined threshold is indicative of the subject being afflicted with a moderate form of PD; and (3) the determined level of the at least one RNA polynucleotide sequence listed under any one of Tables 1, 3, and 4 being greater than the pre-determined threshold or the determined level of the at least one RNA polynucleotide sequence listed under Table 2 being lower than the pre-determined threshold is indicative of the subject being afflicted with a mild form of PD, thereby determining the severity of PD in the subject afflicted therewith.

[0013] According to another aspect, there is provided a method for determining responsiveness to anti PD therapy of a subject administered therewith, the method comprising determining in a sample obtained or derived from a subject administered with an anti PD therapy a level of at least one RNA polynucleotide sequence listed under any one ofTables 1-4, wherein an increased level of at least one RNA polynucleotide sequence listed under any one of Tables 1, 3, and 4 in the sample compared to a control, a decreased level of at least one RNA polynucleotide sequence listed under Table 2 in the sample compared to a control, or both, is indicative of the subject being responsive to the anti PD therapy, thereby determining responsiveness to anti PD therapy of the subject administered therewith.

[0001] In some embodiments, modulated is increased or decreased compared to the control.

[0015] In some embodiments, modulated is decreased compared to the control, and the at least one RNA polynucleotide sequence is listed under any one of Tables 1, 3, and 4.

[0016] In some embodiments, modulated is increased compared to the control, and the at least one RNA polynucleotide sequence is listed under any one of Table 2.

[0017] In some embodiments, the subject is further afflicted with dementia.

[0018] In some embodiments, modulated is decreased compared to the control, and the at least one RNA polynucleotide sequence is listed under Table 1.

[0019] In some embodiments, modulated is increased compared to the control, and the at least one RNA polynucleotide sequence is listed under Table 2.

[0020] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of anti PD therapy.

[0021] In some embodiments, the determining is repeatedly determining. In some embodiments, repeatedly is periodically.

[0022] In some embodiments, the determining is in a plurality of samples being obtained or derived from the subject in a plurality of sampling events.

[0023] In some embodiments, the sample comprises: whole blood, plasma, brain biopsy, or any combination thereof, of the subject.

[0024] In some embodiments, the determining is in vitro or ex vivo determining.

[0025] In some embodiments, the determining is by a method selected from the group consisting of: RNA-sequencing, quantitative polymerase chain reaction (qPCR), and polynucleotide hybridization.

[0026] In some embodiments, the anti PD therapy comprises deep brain stimulation.

[0027] In some embodiments, the anti PD therapy comprises at least one compound selected from the group consisting of: 1-3,4-dihydroxyphenylalanine (L-DOPA), monoamine oxidase B inhibitor, a DOPA decarboxylase inhibitor, a catechol-O-methyltransferase (COMT) inhibitor, bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, lisuride, and any combination thereof.

[0028] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0029] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0030] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0031] Fig. 1 includes a scheme of non-limiting RNA processing according to the current invention.

[0032] Figs. 2A-2D includes plots showing differential expression (DE) analysis in a blood cohort. (2A) PD (DPD+NDPD) vs. NDC - 188 transcripts DE (47 upregulated, 141 downregulated); (2B) DPD vs. NDC - 117 transcripts DE (30 upregulated, 87 downregulated); (2C) NDPD vs. NDC - 161 transcripts DE (23 upregulated, 138downregulated); and (2D) DPD vs. NDPD - 21 transcripts DE (16 upregulated, 5 downregulated).Legend: • = non-significant (NS); * = 1 < log2 Fold Change < -1; • = p. value < 0.05; and » = both.

[0033] Figs. 3A-3D includes plots and a scheme, showing DE analysis in brain cohort. (3A) Substantia nigra (SN) - 60 transcripts DE (28 upregulated, 32 downregulated); (3B) Amygdala (Amy) - 46 transcripts DE (9 upregulated, 37 downregulated); (3C) Middle temporal gyrus (MTG) - 11 transcripts DE (5 upregulated, 6 downregulated). The locations of the brain regions analyzed in 3A-3C are depicted in (3D). Legend: • = NS;s= 1 < log2 Fold Change < -1; • = p. value < 0.05; and " = both.

[0034] Figs. 4A-4D include dendrograms, heatmaps, and a plot. (4A) samples expression clusters in comparison to clinical traits in the form of heatmap (white = low value, red = high value, and grey = missing value). (4B) Clustering dendrogram of dynamic modules of genes, before and after merging of similar expression profiles. (4C) Association of merged modules and clinical traits calculated using Pearson's r method. (4D) Scatterplot of gene significance for diagnosis vs. module membership in the cyan module.

[0035] Figs. 5A-5C include violin plots showing the top 5 significantly DE transcripts in each category. (5A) Top 5 DE transcripts that were shared in the two comparisons, (5B) top 5 DE transcripts that were unique to the PDD vs. NDC comparison and (5C) top 5 DE transcripts that were unique to the NDPD vs. NDC comparison.

[0036] Figs. 6A-6D include graphs showing correlation between cholinergic related genes using Pearson method. On the right top corner of each graph are the absolute values of the correlation test with the significance level in red asterisks. On the left bottom corner of each graph are the bivariate scatterplots with fitted lines. p-values(0, 0.001, 0.01, 0.05, 0.1, 1) <=> symbols (“***”, " “) (6A) AChE and BChE correlated with top 5 shared transcripts in the NDC group, (6B) AChE and BChE correlated with top 5 transcripts unique to the NDPD comparison in the NDC group, (6C) AChE and BChE correlated with top 5 transcripts unique to the PDD comparison in the NDC group and (6D) in the NDPD group.

[0037] Figs. 7A-7C include a dot expression plot and boxplots showing sex-related DE transcripts. (7A) Dot expression plot of DE expressed x-linked transcripts acrosscomparisons. Dots of size 1 or larger are statisticly significant. (7B) Boxplot of AC004554.2 and (7C) boxplot of QRICH2.DETAILED DESCRIPTION

[0038] According to a first aspect, there is provided a method for diagnosing Parkinson’s disease (PD) in a subject.

[0039] According to another aspect, there is provided a method for diagnosing PD with dementia in a subject.

[0040] According to another aspect, there is provided a method for diagnosing PD without dementia in a subject.

[0041] According to another aspect, there is provided a method for determining the severity of PD in a subject afflicted therewith.

[0042] According to another aspect, there is provided a method for determining responsiveness to anti PD therapy of a subject administered therewith.

[0043] As used herein, the terms “Parkinson’s disease” or “PD” refer to a progressive neurodegenerative disorder characterized primarily by motor symptoms such as tremors, rigidity, bradykinesia (slowness of movement), and postural instability. It is caused by the loss of dopamine -producing neurons in the substantia nigra, a region of the brain that plays a key role in movement control. In addition to motor symptoms, PD can also present nonmotor symptoms including cognitive impairment, mood disorders, sleep disturbances, autonomic dysfunction, and pain. The exact cause of Parkinson’s disease is not fully understood, but it is believed to involve a combination of genetic, environmental, and other factors. Current treatments focus on managing symptoms, primarily through dopamine replacement therapies and, in some cases, surgical interventions such as deep brain stimulation.

[0044] In some embodiments, the method comprises determining in a sample obtained or derived from the subject a level of at least one RNA polynucleotide sequence listed under any one of Tables 1-5.

[0045] In some embodiments, the method comprises determining in a sample obtained or derived from the subject a level of at least one RNA polynucleotide sequence listed under Table 1. In some embodiments, Table 1 lists RNA polynucleotide sequences that are differentially expressed in subjects with PD with dementia (DPD) vs. non-demented controls (NDC). In some embodiments, Table 1 lists RNA polynucleotide sequences that are differentially expressed only in subjects with PD with dementia (DPD) vs. non-demented controls (NDC). In some embodiments, RNA polynucleotide sequences listed in Table 1 are not differentially expressed in non-demented PD subjects (NDPD) vs. non-demented controls (NDC). In some embodiments, Table 1 lists RNA polynucleotide sequences that are down-regulated (e.g., having reduced expression) in subjects with PD with dementia (DPD) vs. non-demented controls (NDC). In some embodiments, Table 1 lists RNA polynucleotide sequences that are down-regulated only in subjects with PD with dementia (DPD) vs. nondemented controls (NDC). In some embodiments, RNA polynucleotide sequences listed in Table 1 are not down-regulated in non-demented PD subjects (NDPD) vs. non-demented controls (NDC). In some embodiments, The RNA polynucleotide sequence listed under Table 1, comprises a nucleic acid sequence set forth in SEQ ID Nos: 1-9, or a functional analog thereof.Table 1. Transcripts that are down-regulated in PD with dementia (DPD) vs. nondemented controls (NDC).

[0046] In some embodiments, the method comprises determining in a sample obtained or derived from the subject a level of at least one RNA polynucleotide sequence listed under Table 2. In some embodiments, Table 2 lists RNA polynucleotide sequences that are differentially expressed in subjects with PD with dementia (DPD) vs. non-demented controls (NDC). In some embodiments, Table 2 lists RNA polynucleotide sequences that are differentially expressed only in subjects with PD with dementia (DPD) vs. non-demented controls (NDC). In some embodiments, RNA polynucleotide sequences listed in Table 2 are not differentially expressed in non-demented PD subjects (NDPD) vs. non-demented controls (NDC). In some embodiments, Table 2 lists RNA polynucleotide sequences that are upregulated (e.g., having increased expression) in subjects with PD with dementia (DPD) vs. non-demented controls (NDC). In some embodiments, Table 2 lists RNA polynucleotide sequences that are upregulated only in subjects with PD with dementia (DPD) vs. nondemented controls (NDC). In some embodiments, RNA polynucleotide sequences listed in Table 2 are not upregulated in non-demented PD subjects (NDPD) vs. non-demented controls (NDC). In some embodiments, The RNA polynucleotide sequence listed under Table 2, comprises a nucleic acid sequence set forth in SEQ ID Nos: 10-15, or a functional analog thereof.Table 2. Transcripts that are upregulated in PD with dementia (DPD) vs. nondemented controls (NDC).

[0047] In some embodiments, the method comprises determining in a sample obtained or derived from the subject a level of at least one RNA polynucleotide sequence listed under Table 3. In some embodiments, Table 3 lists RNA polynucleotide sequences that are differentially expressed in subjects with PD without dementia (NDPD) vs. non-demented controls (NDC). In some embodiments, Table 3 lists RNA polynucleotide sequences that are differentially expressed only in subjects with PD without dementia (NDPD) vs. nondemented controls (NDC). In some embodiments, RNA polynucleotide sequences listed in Table 3 are not differentially expressed in PD subjects with dementia (DPD) vs. nondemented controls (NDC). In some embodiments, Table 3 lists RNA polynucleotide sequences that are down-regulated (e.g., having reduced expression) in subjects with PD without dementia (NDPD) vs. non-demented controls (NDC). In some embodiments, Table 3 lists RNA polynucleotide sequences that are down-regulated only in subjects with PD without dementia (NDPD) vs. non-demented controls (NDC). In some embodiments, RNA polynucleotide sequences listed in Table 3 are not down-regulated in PD subjects with dementia (DPD) vs. non-demented controls (NDC). In some embodiments, The RNA polynucleotide sequence listed under Table 3, comprises a nucleic acid sequence set forth in SEQ ID Nos: 16-25, or a functional analog thereof.Table 3. Transcripts that are down-regulated in PD without dementia (NDPD) vs. nondemented controls (NDC).

[0048] In some embodiments, the method comprises determining in a sample obtained or derived from the subject a level of at least one RNA polynucleotide sequence listed under Table 4. In some embodiments, Table 4 lists RNA polynucleotide sequences that are differentially expressed in subjects with PD without dementia (NDPD) and with dementia (DPD) vs. non-demented controls (NDC). In some embodiments, Table 4 lists RNA polynucleotide sequences that are down-regulated (e.g., having reduced expression) in subjects with PD without dementia (NDPD) and with dementia (DPD) vs. non-demented controls (NDC). In some embodiments, The RNA polynucleotide sequence listed under Table 4, comprises a nucleic acid sequence set forth in SEQ ID Nos: 26-35, or a functional analog thereof.Table 4. Transcripts that are down-regulated in PD without dementia (NDPD) and with dementia (DPD) vs. non-demented controls (NDC).

[0049] In some embodiments, the method comprises determining in a sample obtained or derived from the subject a level of at least one RNA polynucleotide sequence listed under Table 5. In some embodiments, Table 5 lists RNA polynucleotide sequences that are chromosome X linked and / or sex related, and are differentially expressed in subjects with PD without dementia (NDPD) vs. non-demented controls (NDC), in PD subjects with dementia (DPD) vs. non-demented controls (NDC), or both. In some embodiments, Table 5 lists RNA polynucleotide sequences that are down-regulated (e.g., having reduced expression) or upregulated in subjects with PD without dementia (NDPD) vs. non-demented controls (NDC), in PD subjects with dementia (DPD) vs. non-demented controls (NDC), or both. In some embodiments, The RNA polynucleotide sequence listed under Table 5, comprises a nucleic acid sequence set forth in SEQ ID Nos: 4, 13, and 36-45, or a functional analog thereof.Table 5. Transcripts that are chromosome X linked or otherwise sex related, and are differentially expressed in PD subjects without dementia (NDPD) vs. non-demented controls (NDC), in PD subjects with dementia (DPD) vs. non-demented controls (NDC), or both.

[0050] In some embodiments, the method comprises determining in a sample obtained or derived from the subject a level of at least one RNA polynucleotide sequence listed under Table 1, Table 2, or both.

[0051] The term "functional analog" refers to a molecule that, although not necessarily identical in structure to another molecule, performs a similar function or has similar biological activity. In the context of RNA polynucleotide sequences, a functional analog would be a sequence that, while not identical to the reference sequence, can still perform the same or similar biological function, such as binding to the same target or participating in the same cellular process. In some embodiments, a functional analog is a fragment of an RNA polynucleotide sequence listed under any one of Tables 1-5. In some embodiments, a functional analog is a fragment of an RNA polynucleotide sequence having a nucleic acid sequence set forth in SEQ ID Nos: 1-45. In some embodiments, the fragment is identical tothe RNA polynucleotide sequence listed under any one of Tables 1-5, and is shorter thereof. In some embodiments, the fragment comprises at least 1, 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 800, 900, or 1,000 less nucleotides compared to the RNA polynucleotide listed under any one of Tables 1-5, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the fragment is shorter than the RNA polynucleotide listed under any one of Tables 1-5 by at least 1, 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 800, 900, or 1,000, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the length of the fragment is sufficient for primers annealing thereto, probe hybridization thereto, or both.

[0052] Parameters required for PCR amplification, primers annealing, probe hybridization, or any combination thereof, are common and would be apparent to one of ordinary skill in the art. Exemplary teachings can be identified in Molecular Cloning: A laboratory Manual" Sambrook et al., (1989).

[0053] In some embodiments, the method comprises determining in a sample obtained or derived from the subject a level of at least one RNA polynucleotide sequence listed under Table 3.

[0054] In some embodiments, the method comprises determining in a sample obtained or derived from the subject a level of at least one RNA polynucleotide sequence listed under any one of Tables 1-4; and comparing the determined level of the at least one RNA polynucleotide sequence to a pre-determined threshold.

[0055] In some embodiments, the method comprises determining in a sample obtained or derived from a subject administered with an anti PD therapy a level of at least one RNA polynucleotide sequence listed under any one of Tables 1-4. In some embodiments, the subject is administered with a therapeutically effective amount of the anti PD therapy. In some embodiments, the subject is administered with the anti PD therapy so as to treat or alleviate at least one symptom of PD.

[0056] In some embodiments, a modulated level of at least one RNA polynucleotide sequence compared to a control, is indicative of the subject being afflicted with PD.

[0057] In some embodiments, modulated is increased or decreased. In some embodiments, increased or decreased is compared to a control.

[0058] In some embodiments, a control is a healthy control subject. In some embodiments, a control comprises a sample obtained or derived from a healthy subject. In some embodiments, a control is a non-demented PD subject (NDPD). In some embodiments, a control comprises a sample obtained or derived from a non-demented PD subject (NDPD). In some embodiments, a control is a PD subject with dementia (DPD). In some embodiments, a control comprises a sample obtained or derived from a PD subject with dementia (DPD). In some embodiments, a control comprises a sample obtained or derived from a PD subject before administration of anti PD therapy. In some embodiments, a control comprises a sample obtained or derived from a PD subject with dementia (DPD) before administration of anti PD therapy. In some embodiments, a control comprises a sample obtained or derived from a non-demented PD subject (NDPD) before administration of anti PD therapy. In some embodiments, a control comprises a sample obtained or derived from any tissue excluding whole blood, plasma, brain biopsy, or any combination thereof, of a subject. In some embodiments, a control comprises RNA of a subject. In some embodiments, a control comprises RNA obtained, derived, or extracted from any tissue excluding whole blood, plasma, brain biopsy, or any combination thereof, of a subject.

[0059] In some embodiments, greater or increased comprises an increase of at least 2%, 5%, 10%, 15%, 25%, 35%, 50%, 70%, 85%, 90%, 95%, 100%, 200%, 350%, 500%, 750%, 1,000%, or any value and range therebetween, compared to control. Each possibility represents a separate embodiment of the invention.

[0060] In some embodiments, reduced comprises a reduction of at least 2%, 5%, 10%, 15%, 25%, 35%, 50%, 70%, 85%, 90%, 95%, 100%, or any value and range therebetween, compared to control. Each possibility represents a separate embodiment of the invention.

[0061] In some embodiments, modulated or modulation is a decrease compared to a control, and at least one RNA polynucleotide sequence is listed under any one of Tables 1, 3, and 4. In some embodiments, modulated is decreased compared to a control, and at least one RNA polynucleotide sequence comprises a nucleic acid sequence set forth in SEQ ID Nos: 1-9, and 16-35.

[0062] In some embodiments, modulated or modulation is an increase compared to a control, and at least one RNA polynucleotide sequence is listed under any one of Table 2. In some embodiments, modulation is an increase compared to a control, and at least one RNA polynucleotide sequence comprises a nucleic acid sequence set forth in SEQ ID Nos: 10-15.

[0063] In some embodiments, a modulated level of at least one RNA polynucleotide sequence compared to a control, is indicative of the subject being afflicted with PD with dementia. In some embodiments, a modulated level of at least one RNA polynucleotide sequence compared to a control, is indicative of the subject being at increased risk of developing PD with dementia, compared to a control. In some embodiments, a modulated level of at least one RNA polynucleotide sequence compared to a control, is indicative of the subject having predisposition or increased likelihood to developing PD with dementia, compared to a control. In some embodiments, a level of at least one RNA polynucleotide sequence being essentially similar or identical to a control, is indicative of the subject not being afflicted with PD with dementia.

[0064] In some embodiments, modulated or modulation is a decrease compared to a control, and at least one RNA polynucleotide sequence is listed under Table 1. In some embodiments, modulated or modulation is a decrease compared to a control, and at least one RNA polynucleotide sequence comprises a nucleic acid sequence set forth in SEQ ID Nos: 1-9.

[0065] In some embodiments, modulated or modulation is an increase compared to a control, and at least one RNA polynucleotide sequence is listed under Table 2. In some embodiments, modulated or modulation is an increase compared to a control, and at least one RNA polynucleotide sequence comprises a nucleic acid sequence set forth in SEQ ID Nos: 10-15.

[0066] In some embodiments, a reduced level of at least one RNA polynucleotide sequence compared to a control, is indicative of a subject being afflicted with PD without dementia. In some embodiments, a reduced level of at least one RNA polynucleotide sequence compared to a control, is indicative of the subject being at increased risk or prospect of developing PD without dementia, compared to a control. In some embodiments, a reduced level of at least one RNA polynucleotide sequence compared to a control, is indicative of the subject having predisposition or increased likelihood to developing PD without dementia, compared to a control. In some embodiments, a level of at least one RNApolynucleotide sequence, being essentially similar or identical to a control, is indicative of the subject not being afflicted with PD without dementia.

[0067] In some embodiments, a determined level of at least one RNA polynucleotide sequence listed under any one of Tables 1, 3, and 4 being lower than a pre-determined threshold is indicative of a subject being afflicted with an immoderate form of PD. In some embodiments, a determined level of at least one RNA polynucleotide sequence comprising a nucleic acid sequence set forth in SEQ ID Nos: 1-9, and 16-35 being lower than a predetermined threshold is indicative of a subject being afflicted with an immoderate form of PD.

[0068] In some embodiments, a determined level of at least one RNA polynucleotide sequence listed under Table 2 being greater than a pre-determined threshold is indicative of a subject being afflicted with an immoderate form of PD. In some embodiments, a determined level of at least one RNA polynucleotide sequence comprising a nucleic acid sequence set forth in SEQ ID Nos: 10-15 being greater than a pre-determined threshold is indicative of a subject being afflicted with an immoderate form of PD.

[0069] In some embodiments, a determined level of at least one RNA polynucleotide sequence listed under any one of Tables 1, 3, and 4 being greater than a pre-determined threshold is indicative of a subject being afflicted with a mild form of PD. In some embodiments, a determined level of at least one RNA polynucleotide sequence comprising a nucleic acid sequence set forth in SEQ ID Nos: 1-9, and 16-35 being greater than a predetermined threshold is indicative of a subject being afflicted with a mild form of PD.

[0070] In some embodiments, a determined level of at least one RNA polynucleotide sequence listed under any one of Table 2 being lower than a pre-determined threshold is indicative of a subject being afflicted with a mild form of PD. In some embodiments, a determined level of at least one RNA polynucleotide sequence comprising a nucleic acid sequence set forth in SEQ ID Nos: 10-15 being lower than a pre-determined threshold is indicative of a subject being afflicted with a mild form of PD.

[0071] In some embodiments, an increased level of at least one RNA polynucleotide sequence listed under any one of Tables 1, 3, and 4 in the subject or a sample obtained or derived therefrom compared to a control is indicative of the subject being responsive to ananti PD therapy. In some embodiments, an increased level of at least one RNA polynucleotide sequence comprising a nucleic acid sequence set forth in SEQ ID Nos: 1-9, and 16-35 in the subject or a sample obtained or derived therefrom compared to a control is indicative of the subject being responsive to an anti PD therapy.

[0072] In some embodiments, an equal, equivalent, or reduced level of at least one RNA polynucleotide sequence listed under any one of Tables 1, 3, and 4 in the subject or a sample obtained or derived therefrom compared to a control is indicative of the subject being non- responsive to an anti PD therapy. In some embodiments, an equal, equivalent, or reduced level of at least one RNA polynucleotide sequence comprising a nucleic acid sequence set forth in SEQ ID Nos: 1-9, and 16-35 in the subject or a sample obtained or derived therefrom compared to a control is indicative of the subject being non-responsive to an anti PD therapy.

[0073] In some embodiments, an equal, equivalent, or increased level of at least one RNA polynucleotide sequence listed under Table 2 in the subject or a sample obtained or derived therefrom compared to a control, or both, is indicative of the subject being non-responsive to an anti PD therapy. In some embodiments, an equal, equivalent, or increased level of at least one RNA polynucleotide sequence comprising a nucleic acid sequence set forth in SEQ ID Nos: 10-15 in the subject or a sample obtained or derived therefrom, compared to a control is indicative of the subject being non-responsive to an anti PD therapy.

[0074] In some embodiments, a decreased level of at least one RNA polynucleotide sequence listed under Table 2 in the subject or a sample obtained or derived therefrom compared to a control, or both, is indicative of the subject being responsive to an anti PD therapy. In some embodiments, a decreased level of at least one RNA polynucleotide sequence comprising a nucleic acid sequence set forth in SEQ ID Nos: 10-15 in the subject or a sample obtained or derived therefrom compared to a control is indicative of the subject being responsive to an anti PD therapy.

[0075] In some embodiments, the determining comprises repeatedly determining. In some embodiments, repeatedly comprises periodically.

[0076] In some embodiments, the determining is in a plurality of samples. In some embodiments, the plurality of samples is obtained or derived from the subject in a plurality of sampling events.

[0077] As used herein, the term "plurality" refers to any integer being equal to or greater than 2.

[0078] In some embodiments, a sample comprises: whole blood, plasma, brain biopsy, or any combination thereof, of a subject. In some embodiments, a sample comprises RNA obtained, derived, or extracted from whole blood, plasma, brain biopsy, or any combination thereof, of a subject.

[0079] In some embodiments, determining is in vitro or ex vivo determining.

[0080] In some embodiments, determining is by a method selected from: RNA-sequencing, quantitative polymerase chain reaction (qPCR), polynucleotide hybridization, or any combination thereof.

[0081] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of anti PD therapy.

[0082] As used herein, the term “anti PD therapy” encompasses any procedure, stimulation, drug, etc., which at least alleviates one or more symptoms of PD.

[0083] Compounds and means for alleviating PD symptoms would be apparent to one of ordinary skill in the art, such as further specified herein below.

[0084] In some embodiments, anti PD therapy comprises deep brain stimulation.

[0085] In some embodiments, anti PD therapy comprises at least one compound selected from: 1-3,4-dihydroxyphenylalanine (L-DOPA), monoamine oxidase B inhibitor, a DOPA decarboxylase inhibitor, a catechol-O-methyltransferase (COMT) inhibitor, bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, lisuride, or any combination thereof.

[0086] In some embodiments, a pre-determined threshold is a range of pre-determined thresholds. In some embodiments, a pre-determined threshold is a value with a standard deviation, standard error, or both. In some embodiments, a pre-determined threshold is a value ± 1-20%, 1-5%, 1-10%, 1-15%, 5-10%. 5-15%, or 5-20%. Each possibility represents a separate embodiment of the invention.

[0087] In some embodiments, determining is in a plurality of samples being obtained or derived from the subject. In some embodiments, the plurality of samples are obtained, collected, sampled, or any equivalent thereof, from the subject in a plurality of sampling events. In some embodiments, the plurality of sampling events are at least 1 day, 3 days, 5 days, 7 days, 14 days, 30 days, 2 months, 3 months, 5 months, 6 months, 8 months, 10 months, or 1 year apart, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0088] In some embodiments, at least one second sample of a plurality of samples is obtained from the subject chronologically after or later then the first sample of the plurality of samples.

[0089] In some embodiments, the method further comprises a step preceding or before the determining step, comprising obtaining a sample from the subject.

[0090] In some embodiments, the method utilized polynucleotide amplification. In some embodiments, amplification is or comprises polymerase chain reaction (PCR).

[0091] In some embodiments, the method utilizes polynucleotide hybridization.

[0092] In some embodiments, PCR comprises denaturing double- stranded DNA in a sample (to separate the complementary strands), annealing the primers to the dissociated DNA strands, and extension reaction from the primers catalyzed by a thermostable DNA polymerase, the cycle is then repeated.

[0093] In some embodiments, the type and / or regimen of an anti PD therapy is selected from: type of drug, dose of drug, frequency of administration, DBS application, frequency and / or intensity of DBS, or any combination thereof.

[0094] In some embodiments, mild form of PD comprises or is characterized by low symptom(s) of PD.

[0095] As used herein, the term "mild form of PD" refers to a stage of Parkinson's disease characterized by low or minimal symptoms. In this stage, the symptoms of PD are present but are not severe enough to significantly impact the daily activities and quality of life of the patient. The symptoms may include slight tremors, minor rigidity, or mild bradykinesia (slowness of movement), but the patient can generally manage their routine tasks with littleto no assistance. This stage is often the initial phase and / or indicator of the disease, where early diagnosis and intervention can be crucial for managing the progression of PD.

[0096] As used herein, the term "immoderate form of PD" refers to a stage of Parkinson's disease where the symptoms are more pronounced than in the mild form but not as severe as in the advanced stages. In this stage, the symptoms of PD, such as tremors, rigidity, bradykinesia (slowness of movement), and postural instability, are more noticeable and begin to interfere with daily activities and quality of life. Patients may require some assistance with routine tasks and may experience increased difficulty in movement and coordination. This stage represents a progression from the mild form and indicates a need for more intensive management and treatment to control the symptoms and slow the disease's progression.

[0097] As used herein, the terms “treatment” or “treating” of a disease, disorder or condition (e.g., PD) encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment may be palliative in nature and need not mean that the disease, disorder or condition is totally or partially cured. To be an effective treatment, a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.

[0098] In some embodiments, treating comprises preventing appearance and / or onset and / or development of at least one symptom associated with a disease, disorder or condition (e.g., PD), severity thereof, or inhibition of the progression thereof.

[0099] According to another aspect, there is provided a method for detecting a modulated level of two or more RNA polynucleotide sequences listed under any one of Tables 1-5 in a subject, compared to a control.

[0100] In some embodiments, the method comprises: (a) obtaining a sample from the subject; and (b) determining whether the two or more RNA polynucleotide sequences listed under any one of Tables 1-5 are present in a modulated level compared to a control in the sample by contacting the sample with two or more exogenous or synthetic polynucleotides being complementary to the two or more RNA polynucleotide sequences, and determininghybridization between the two or more RNA polynucleotide and the two or more exogenous or synthetic polynucleotides.

[0101] According to another aspect, there is provided a method for diagnosing PD in a subject, the method comprising: (a) obtaining a sample from the subject; (b) determining whether two or more RNA polynucleotide sequences listed under any one of Tables 1-5 are present in a modulated level compared to a control in the sample by contacting the sample with two or more exogenous or synthetic polynucleotides being complementary to the two or more RNA polynucleotide sequences, and determining hybridization between the two or more RNA polynucleotide and the two or more exogenous or synthetic polynucleotides; and (c) diagnosing the subject with PD when the presence of a modulated level of the two or more RNA polynucleotide sequences listed under any one of Tables 1-5, compared to a control, is determined.

[0102] In some embodiments, the two or more exogenous or synthetic polynucleotides are probes and / or primers.

[0103] In some embodiments, primers are primer pair for PCR amplification. Methods, means, and considerations for designing primers suitable for PCR amplification, as well as probes, are common and would be highly apparent to a person of ordinary skill in the art, such as, but not limited, to Primer3web, and Primer-BLAST designing tools.

[0104] Types of nucleic acid amplification are common and would be apparent to one of ordinary skill in the art. Non-limiting examples for such methods include, but are not limited to, Quantitative PCR (qPCR), Reverse transcription PCR (RT-PCR), Loop-mediated isothermal amplification (LAMP), Nucleic acid sequence -based amplification (NASBA), Rolling circle amplification (RCA), Helicase-dependent amplification (HD A), Strand displacement amplification (SDA), Multiple displacement amplification (MDA), Transcription-mediated amplification (TMA), Ligase chain reaction (LCR), Branched DNA (bDNA) amplification.

[0105] In some embodiments, the method further comprises a reverse transcription step comprising synthesizing complementary DNA (cDNA) based on the RNA in the sample.

[0106] In some embodiments, probes are detectable probes. In some embodiments, probes comprise a detectable moiety. In some embodiments, probes are nucleic acid-based probes.

[0107] The term "nucleic acid-based probes" refers to probes that are composed of nucleic acids, such as DNA or RNA, which are designed to hybridize specifically to complementary sequences of nucleic acids in a sample. These probes are used in various molecular biology techniques to detect, quantify, or isolate specific nucleic acid sequences. They often contain a detectable moiety, such as a fluorescent dye, radioactive label, or enzyme, which allows for the visualization or measurement of the hybridization event.

[0108] Molecular methods utilizing nucleic acid-based probes are common and would be apparent to one of ordinary skill in the art. Non-limiting examples for such methods include, but are not limited to, Fluorescence in situ hybridization (FISH), Polymerase chain reaction (PCR), Quantitative PCR (qPCR), Reverse transcription PCR (RT-PCR), Southern blotting, Northern blotting, Microarray analysis, Next-generation sequencing (NGS), Dot blot hybridization, In situ hybridization (ISH), DNA / RNA sequencing, and CRISPR-based detection methods.

[0109] According to another aspect, there is provided a kit comprising 2 to 150 exogenous or synthetic polynucleotides being complementary to two or more RNA polynucleotide sequences listed under any one of Tables 1-5. In some embodiments, the kit further comprises instructions for determining the amount or abundance of two or more RNA polynucleotide sequences listed under any one of Tables 1-5 in a sample. In some embodiments, the 2 to 150 exogenous or synthetic polynucleotides are probes, primers, or both. In some embodiments, any one of the 2 to 150 exogenous or synthetic polynucleotides has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% complementarity to an equal-length portion of an RNA polynucleotide sequence listed under any one of Tables 1-5 (or SEQ ID Nos: 1-45), or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, any one of the 2 to 150 exogenous or synthetic polynucleotides has 70-80%, 75-85%, 80-90%, 85-95%, 90-99%, or 95-100% complementarity to an equal-length portion of an RNA polynucleotide sequence listed under any one of Tables 1-5 (or SEQ ID Nos: 1-45). Each possibility represents a separate embodiment of the invention.

[0110] In some embodiments, the kit is for use in a method for diagnosing PD, diagnosing PD with dementia, diagnosing PD without dementia, determining responsiveness to anti PD therapy of a subject administered therewith, determining the severity of PD in a subject afflicted therewith, or any combination thereof.

[0111] As used herein, "treating" comprises ameliorating, preventing, or both.

[0112] As used herein, the terms “administering”, “administration”, and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect. One aspect of the present subject matter provides for dermal or transdermal administration of a therapeutically effective amount of a composition of the present subject matter to a subject in need thereof. Other suitable routes of administration can include oral, dermal, transdermal, parenteral, subcutaneous, intravenous, intramuscular, or intraperitoneal. In some embodiments, the administering is systemic administering. In some embodiments, the administering is to the inflamed skin site.

[0113] In some embodiments, compositions for use in the methods of this invention comprise solutions or emulsions, which in some embodiments are aqueous solutions or emulsions comprising a safe and effective amount of the cannabinoid of the present invention and optionally, other compounds as described herein.

[0114] In some embodiments, the composition is formulated with a carrier. In some embodiments, the composition is encapsulated.

[0115] In one embodiment, the amount of a composition to be administered will be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0116] The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0117] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that statedrange, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0118] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1,000 nucleobases refers to a length of 1,000 nucleobases ± 100 nucleobases.

[0119] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0120] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.

[0121] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a”, “an” and “at least one” are used interchangeably in this application.

[0122] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0123] In the description and claims of the present application, each of the verbs, “comprise”, “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.

[0124] Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0125] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive.

[0126] Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising,” indicate the inclusion of any recited integer or group of integers but not the exclusion of any other integer or group of integers.

[0127] As used herein, the term “consists essentially of’, or variations such as “consist essentially of’ or “consisting essentially of’ as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition.

[0128] As used herein, the terms "comprises", "comprising", "containing", "having" and the like can mean "includes", "including", and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. In one embodiment, the terms "comprises", "comprising", "having" are / is interchangeable with "consisting".

[0129] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.EXAMPLES

[0130] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include chemical, molecular, biochemical, computational, statistics and cell biology techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); The Organic Chemistry of Biological Pathways by John McMurry and Tadhg Begley (Roberts and Company, 2005); Organic Chemistry of Enzyme-Catalyzed Reactions by Richard Silverman (Academic Press, 2002); Organic Chemistry (6thEdition) by Leroy "Skip" G Wade; Organic Chemistry by T. W. Graham Solomons and, Craig Fryhle.Material and MethodsSamples collection and RNA preparation

[0131] Frozen human brain tissues and postmortem whole blood samples were obtained from the Netherlands Brain Bank (NBB; Amsterdam, The Netherlands) and approved for use by the Netherlands Brain Bank and by the Ethics Committee of the Hebrew University. Three groups of blood samples were available - from demented and non-demented PD patients (DPD, NDPD) and non-demented controls (NDC), as well as two groups of brain samples (DPD and NDC). Samples were disqualified if they were derived from PD patients with Alzheimer comorbidity, deep brain stimulation or unknown cause of death.Table 6. Separation to patient groups in each cohortBlood samples

[0132] Whole blood samples were collected within an average of 4:45 hours post-mortem (SD = 01:46 hours) and kept frozen at -80 °C until use. The NucleoSpin RNA Blood kit served for RNA extraction according to manufacturer’s instructions. Briefly, blood samples were defrosted in the presence of an equal amount of lysis buffer, then proteinase K was added to the mixture and the tubes were incubated at room temperature for 15 minutes while being vigorously shaken on a tubes’ shaker. Then an equal amount of ethanol 70% was added to the mixture and the tubes were centrifuged shortly. Afterwards, the mixture was uploaded to columns and centrifuged for 30 seconds at 11,000 g in batches of 600 pL. Next, a membrane desalting buffer was added, followed by another step of similar centrifugation. Later, DNase was added to digest DNA, and the mixture was incubated at room temperature for 15 minutes, followed by three steps of buffer wash and centrifugation to wash and dry the silica membrane. Finally, the RNA was eluted with 60 p L RNase-free H2O.Brain samples

[0133] Frozen human brain tissues from the Substantia nigra (SN), Amygdala (Amy) and medial temporal gyrus (MTG) were collected within an average of 6:30 hours (SD = 2:20 hours) from death. For RNA extraction, tissue pieces cut on dry ice were snap-frozen in liquid nitrogen to maintain RNA integrity. Lysis and homogenization (using a pellet pestle) were performed with 700 ml Qiazol Lysis Reagent, after which homogenates were re-snap- frozen, and RNA extraction with the miRNeasy Kit (both from Qiagen, Germantown, MD, USA) followed.RNA quality checks

[0134] RNA concentration and quality were measured using Nanodrop and electrophoresis in 1% agarose gels. Samples with degraded RNA were disqualified, and the rest were subjected to Bioanalyzer analysis for assessing RNA integrity number (RIN). Average RIN values were 6.21±1.07 for blood samples, and 7.25±0.64 for brain samples. Sadly, only a few female blood samples passed the quality checks, too few to enable statistical significance analysis, therefore the inventors continued the analysis on male samples alone, in both the brain and blood cohorts.RNA sequencing and data extraction

[0135] The samples which passed the previous quality tests were sent to long RNA (maximum read length of 80 bp), single-end sequencing using an Illumine TrueSeq kit, according to the manufacturer's instructions. Every flow cell had 8 samples and the average reads per sample were -50,000 million. RNA-seq data quality was measured using the FastQC program, leading to disqualification of a few more samples. The fastq files were cleaned from rRNA (average of 5%) and were aligned to the UCSC hg38 genome (the Genome Reference Consortium Human GRCh38.pl4 (GCA_000001405.29)) using STAR. Countifying reads per gene was done using HTSeq.Differential expression analysis

[0136] Differential expression (DE) analysis was done using the DESeq2 R package in R 3.6.1. HTSeq files were uploaded and combined to create a normalized read count matrix for all genes and samples. DE was calculated for blood and distinct brain regions (SN, Amy, MTG) separately, comparing DPD vs. NDPD, and seeking four comparisons in the blood: (1) all PD patients vs. NDC, (2) DPD vs. NDC, (3) NDPD vs. NDC, and (4) DPD vs. NDPD. All analyses were balanced for Batch effect, RIN and death by sepsis, as well as for multiple testing using FDR.Networks analysis

[0137] The inventors conducted the weighted gene co-expression network analysis (WGCNA) R package to find gene networks demonstrating inter-related changes. First, the normalized count matrix that was created using the countsnorm function from the DESeq2package was uploaded, cleaned and filtered from outliers using clustering dendrogram of the samples based on their Euclidean distance.

[0138] Secondly, the inventors created a gene network and identified the inter-related modules, using the "Step-by-step" option from the WGCNA manual. A soft threshold was chosen based on the " Scale independence" and "Mean Connectivity" plots, at the value of 8. Then the adjacencies were calculated and based on them, the Topological Overlap Matrix (TOM) as well. The TOM was used to produce a hierarchical clustering tree (dendrogram) of genes. The genes were then clustered into modules using the Dynamic Tree Cut method, followed by a further merging of modules whose expression profiles are very similar.

[0139] Third and last, the inventors uploaded a patients' traits file and worked to identify modules that are significantly associated with the patients' traits. Ultimately, the inventors looked more closely at the relationship between the diagnoses of the patients to specific modules by intramodular analysis.Correlation analysis

[0140] To identify the most influential genes to focus on, the inventors compared the significantly differentially expressed genes to a list of cholinergic genes of interest. The ones that were found in both lists were subjected to a correlation test among themselves and with various patients' traits.

[0141] Six (6) samples were disqualified for poor quality after sequencing; leaving 57 samples for further analyses. Out of the PD patients, only 6 were women, 2 in the DPD group and 4 in the NDPD group. Thus, the inventors chose to look only at male patients (total of 38). Also, the NDC group showed a higher age average than the two PD groups, but Kruskal Wallis test revealed that the differences were not significant.EXAMPLE 1Differentially expressed transcripts in brain and blood

[0142] DE profiles in the blood presented distinct expression patterns for the four comparisons (Fig. 2). When comparing all PD patients together as one group (PD = DPD+NDPD) to non-demented controls (NDC), most of the DE transcripts weredownregulated, compatible with previous reports (Fig. 2A). Next, the inventors separately compared demented or non-demented PD patients (NDPD) to the NDC group. Both the DPD vs. NDC and the NDPD vs. NDC comparisons reproduced this pattern (Figs. 2B-2C), with the NDPD vs. NDC showing a more pronounced change.

[0143] However, comparing the DPD to NDPD groups revealed a shifted pattern, presenting upregulation in the majority of the DE blood transcripts from demented compared to the non-demented patients (Fig. 2D). The inventors conclude that the development of dementia in PD patients involves elevated expression of long RNA transcripts in their blood. Yet, looking for group separation using principal components analysis (PCA), t-SNE and heatmap methods failed to reveal clear separation between the three blood group samples, likely due to the small cohort.

[0014] DE patterns in brain tissues between DPD and NDC groups were relatively pronounced in the SN, followed by the stress-responding Amy and lastly the MTG (Figs. 3A-3C, respectively). Surprisingly, the Amy showed similar DE features to the blood patterns, with the majority of the transcripts down regulated (Fig. 3B). The SN and the MTG showed similar numbers of up- and down-regulated transcripts (Figs. 3A and 3C, respectively).EXAMPLE 2Weighted gene co-expression network analysis (WGCNA)

[0145] Data was cleaned and filtered, and samples expression data were plotted alongside clinical traits, showing no specific trait had a mass influence on the samples' clusters (Fig. 4A). Later, TOM was created from adjacencies, producing a hierarchical clustering tree of 30 dynamic modules, which were then cut at height of 0.5 and merged into 15 modules of similar expression profiles (Fig. 4B).

[0146] Next, the inventors associated the merged modules to the clinical traits (Fig. 3C). Three modules were significantly associated with either patients' diagnosis, Braak Lewy bodies (braakLB) score or both. The cyan module was positively correlated with the patients' diagnosis (r=0.41, p.value<0.01), and gene ontology (GO) Enrichment analysisdemonstrated involvement of this module’s transcripts in protein folding and protein heat shock regulation.

[0147] Furthermore, a second module, green-yellow, positively correlated with braaklb score of the tested patients (r=0.35, p.value<0.04) and GO-Enrichment analysis indicated involvement of its transcripts in transforming TGF-beta activation, microvillus assembly and organization and protein geranylgeranylation and prenylation. Lastly, another module, purple, was negatively correlated with both diagnosis and the braaklb (r=-0.34, p.value<0.05 for diagnosis and r=-0.42, p.value<0.01), and GO-Enrichment analysis indicated involvement of its transcripts in cell cycle and DNA replication pathways.EXAMPLE 3Cholinergic gene transcripts correlations

[0148] Finally, the inventors decided to go back to the non-motoric symptoms of PD and look for connections between the DE genes that have been found and the cholinergic system. The inventors identified the top 5 most significantly differentially expressed (DE) transcripts that were shared and unique between the two comparisons (NDPD vs. NDC and DPD vs. NDC, Fig. 5), and tested the correlation between the normalized counts of those transcripts and transcript levels of the acetylcholine hydrolyzing acetyl- and butyrylcholinesterase (AChE, BChE) enzymes in each of the diagnosis groups separately (Fig. 6). The inventors found positive and significant correlations to AChEXBChE in the NDC groups under all conditions, and with the NDPD groups in the top 5 transcripts that were unique to the DPD comparison.EXAMPLE 4Sex-related DE transcripts

[0149] Next, the inventors sought DE transcripts that were shared between both brain and blood datasets. One transcript which matched this criterion, ‘Glutamine-Rich Protein 2’ (QRICH2; Fig. 7C) was upregulated in DPD compared to NDC samples (SN DPD vs. NDC: log2FC = 1.7, p. = 0.014) (blood DPD vs. NDC: log2FC = 1.61, p. = 0.018; PD vs. NDC: log2FC = 1.45, p. = 0.027). QRICH2 acts as a suppressor of ubiquitination and degradationof proteins involved in flagellar development and motility and its expression is surprisingly highest in male testis, where it has an essential role in the formation of sperm flagella and in flagellar structure maintenance.

[0150] Given the finding of QRICH2, the inventors sought other DE sex-specific transcripts by searching for DE X-linked transcripts across all comparisons (Fig. 7A). This search revealed 11 DE transcripts, two in the SN, and the others in one or more of the “blood” comparisons. Specifically, the AC004554.2 long non-coding RNA (IncRNA) transcript (Fig. 7B) which was found to be downregulated in patients with AVF stenosis was upregulated in the demented PD group compared to the non-demented one in the blood (DPD vs. NDPD: log2FC = 3.58, p. = 0.024).

[0151] Of the 10 DE protein coding transcripts, the Shroom Family Member 4 (SHROOM4) transcript is notably linked to a familial subtype of X-linked PD; and Diacyl glycerol Kinase Kappa (DGKK) was shown to regulate a-synuclein protein in-vitro. Intriguingly, these two and three other coding transcripts (ZCCHC12, KCNE5, MIDI) are also related to diverse mental retardation syndromes. Further, the Zinc Finger CCHC Domain-Containing Protein 12 (ZCCHC12) and Retinitis Pigmentosa GTPase Regulator (RPGR) transcripts are related to Muscle deterioration syndromes, and ZCCHC12 also has dopaminergic and cholinergic links along with DGKK. The inventors conclude that X-linked transcripts linked with brain and body diseases show a clear tendency for DE in both blood and brain of PD patients (Fig. 7A).Discussion

[0152] Initial comparison of blood cells transcripts from PD patients with and without dementia to controls without dementia yielded hundreds of DE genes between the groups, showing that long RNA transcripts from whole blood, even collected postmortem, bear significant information for PD diagnosis.

[0153] The inventors identified clusters of transcripts that correlated with patients' diagnosis and Braak Lewy bodies score, using the WGCNA with further GO-enrichment pathway analysis, thus showing a promising signature of biomarkers that may identify not only the disease but also the nature and severity of its symptoms.

[0154] Furthermore, transcript candidates had shown a correlation to cholinesterase transcripts in non-demented controls and in some cases with non-demented PD patients. These findings suggest that the cholinergic system is playing a protective role in those nondemented individuals and may prove significant in the attempt to diagnose them separately.

[0155] Compiled together, the inventors have used these results to identify 4 groups of transcripts that may differentiate non-demented PD patients from demented ones and both groups from non-demented controls. Moreover, many of the currently identified transcript candidates have not been linked to PD before, a result the inventors conclude to be mostly due to the current use of sequencing instead of restricting microarrays.

[0156] The current discovery of a group of X-linked DE transcripts in an all-male cohort strengthens that initiative, and reinforces the importance of pursuing the molecular origin of sex differences in PD. Altogether, the observed sex-linked brain and blood transcripts differentiating between the tested groups largely showed clear links to the PD symptomatology profile, possibly indicating functional relevance.

[0157] In conclusion, the inventors showed here that long RNA transcripts from postmortem brain and whole blood samples hold big promise in the future diagnosis of PD, with and without dementia. Looking deeper into long non-coding RNAs, earlier disease stages, differences between sexes and bigger cohorts may be warranted.

[0158] While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by reference to the claims, which follow.

Claims

CLAIMSWhat is claimed:

1. A method for diagnosing Parkinson’s disease (PD) in a subject, the method comprising determining in a sample obtained or derived from said subject a level of at least one RNA polynucleotide sequence listed under any one of Tables 1-5, wherein a modulated level of said at least one RNA polynucleotide sequence compared to a control, is indicative of said subject being afflicted with PD, thereby diagnosing PD in the subject.

2. The method of claim 1, wherein said modulated is increased or decreased compared to said control.

3. The method of claim 1 or 2, wherein said modulated is decreased compared to said control, and said at least one RNA polynucleotide sequence is listed under any one of Tables1, 3, and 4.

4. The method of claim 1 or 2, wherein said modulated is increased compared to said control, and said at least one RNA polynucleotide sequence is listed under any one of Table2.

5. The method of any one of claims 1 to 4, wherein said subject is further afflicted with dementia.

6. A method for diagnosing PD with dementia in a subject, the method comprising determining in a sample obtained or derived from said subject a level of at least one RNA polynucleotide sequence listed under any one of Tables 1 and 2, wherein a modulated level of said at least one RNA polynucleotide sequence compared to a control, is indicative of said subject being afflicted with PD with dementia, thereby diagnosing PD with dementia in the subject.

7. The method of claim 6, wherein said modulated is increased or decreased compared to said control.

8. The method of claim 6 or 7, wherein said modulated is decreased compared to said control, and said at least one RNA polynucleotide sequence is listed under Table 1.

9. The method of claim 6 or 7, wherein said modulated is increased compared to said control, and said at least one RNA polynucleotide sequence is listed under Table 2.

10. A method for diagnosing PD without dementia in a subject, the method comprising determining in a sample obtained or derived from said subject a level of at least one RNA polynucleotide sequence listed under Table 3, wherein a reduced level of said at least one RNA polynucleotide sequence compared to a control, is indicative of said subject being afflicted with PD without dementia, thereby diagnosing PD without dementia in the subject.

11. A method for determining the severity of PD in a subject afflicted therewith, the method comprising:(i) determining in a sample obtained or derived from said subject a level of at least one RNA polynucleotide sequence listed under any one of Tables 1-4; and(ii) comparing the determined level of the at least one RNA polynucleotide sequence to a pre-determined threshold, wherein any one of: (1) said determined level of said at least one RNA polynucleotide sequence listed under any one of Tables 1, 3, and 4 being lower than said pre-determined threshold or said determined level of said at least one RNA polynucleotide sequence listed under Table 2 being greater than said pre-determined threshold is indicative of said subject being afflicted with an immoderate form of PD; (2) said determined level of said at least one RNA polynucleotide sequence listed under any one of Tables 1-4 being equal to said predetermined threshold is indicative of said subject being afflicted with a moderate form of PD; and (3) said determined level of said at least one RNA polynucleotide sequence listed under any one of Tables 1, 3, and 4 being greater than said pre-determined threshold or said determined level of said at least one RNA polynucleotide sequence listed under Table 2 being lower than said pre-determined threshold is indicative of said subject being afflicted with a mild form of PD, thereby determining the severity of PD in the subject afflicted therewith.

12. A method for determining responsiveness to anti PD therapy of a subject administered therewith, the method comprising determining in a sample obtained or derivedfrom a subject administered with an anti PD therapy a level of at least one RNA polynucleotide sequence listed under any one of Tables 1-4, wherein an increased level of at least one RNA polynucleotide sequence listed under any one of Tables 1, 3, and 4 in said sample compared to a control, a decreased level of at least one RNA polynucleotide sequence listed under Table 2 in said sample compared to a control, or both, is indicative of said subject being responsive to said anti PD therapy, thereby determining responsiveness to anti PD therapy of the subject administered therewith.

13. The method of any one of claims 1 to 12, further comprising administering to said subject a therapeutically effective amount of anti PD therapy.

14. The method of any one of claims 1 to 13, wherein said determining is repeatedly determining.

15. The method of claim 14, wherein said repeatedly is periodically.

16. The method of any one of claims 1 to 15, wherein said determining is in a plurality of samples being obtained or derived from said subject in a plurality of sampling events.

17. The method of any one of claims 1 to 16, wherein sample comprises: whole blood, plasma, brain biopsy, or any combination thereof, of said subject.

18. The method of any one of claims 1 to 17, wherein said determining is in vitro or ex vivo determining.

19. The method of any one of claims 1 to 18, wherein said determining is by a method selected from the group consisting of: RNA-sequencing, quantitative polymerase chain reaction (qPCR), and polynucleotide hybridization.

20. The method of any one of claims 12 to 19, wherein said anti PD therapy comprises deep brain stimulation.

21. The method of any one of claims 12 to 19, wherein said anti PD therapy comprises at least one compound selected from the group consisting of: 1-3,4-dihydroxyphenylalanine (L-DOPA), monoamine oxidase B inhibitor, a DOPA decarboxylase inhibitor, a catechol-O-methyltransferase (COMT) inhibitor, bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, lisuride, and any combination thereof.

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