WO2018008017A1

WO2018008017A1 - Circulating micrornas as biomarkers for therapy in neuromyelitis optica (nmo) and multiple sclerosis (ms)

Info

Publication number: WO2018008017A1
Application number: PCT/IL2017/050741
Authority: WO
Inventors: Iris LAVON BEN MOSHE; Adi VAKNIN-DEMBINSKY
Original assignee: Hadasit Medical Research Services and Development Co
Current assignee: Hadasit Medical Research Services and Development Co
Priority date: 2016-07-03
Filing date: 2017-07-03
Publication date: 2018-01-11
Anticipated expiration: 2019-01-03

Abstract

The invention relates to circulating micro RNAs as biomarkers in Neuromyelitis Optica (NMO) and/or multiple sclerosis (MS). The invention further relates to circulating mi RNA profile for the prediction of responsiveness to treatment, such as, rituximab, of subjects afflicted with NMO and/or MS.

Description

CIRCULATING MICRORNAS AS BIOMARKERS FOR THERAPY IN NEUROMYELITIS OPTICA (NMO) AND MULTIPLE SCLEROSIS

(MS)

FIELD OF THE INVENTION

The invention relates to circulating microRNAs as biomarkers in Neuromyelitis Optica (NMO) and Multiple Sclerosis (MS), for prediction of responsiveness to treatment thereof.

BACKGROUND

Neuromyelitis optica (NMO) is a chronic autoimmune inflammatory disease of the central nervous system (CNS), whose clinical features include mainly acute attacks of bilateral or rapidly sequential optic neuritis (eventually leading to visual loss in many patients), and transverse myelitis. Disease etiology of NMO is still unknown. However, it is known that the inflammatory processes in NMO are mediated by the humoral immune system and primarily target astrocytes [1 , 2]. The most important evidence of this was the identification of the NMO-IgG antibody, anti-Aquaporin-4; NMO-IgG antibodies identify about 82% of patients.

Micro RNAs (miRNAs) function to modify the expression of target genes. miRNA-mediated gene regulation is critical during many biological processes including inflammation and neurodegeneration. Because each miRNA can regulate many target genes, the biological impact of dysregulation of a single miRNA can be considerable. Studies have shown that miRNAs have potential as non-invasive biomarkers for the diagnosis and prognosis of disease as well as monitoring of treatment response [3]; miRNAs also represent promising novel targets for therapy [1]. Extracellular circulating miRNAs are remarkably stable [4]. Their stability is achieved via different mechanisms. They can be packaged in microparticles (exosomes, microvesicles, and apoptotic bodies)[5-7] or associated with RNA- binding proteins (Argonaute2 [Ago2]) or lipoprotein complexes (high-density lipoprotein [HDL]) [8], to prevent their degradation. The stability of miRNAs, coupled with advances in high-throughput technologies that provide the ability to perform a global analysis of miRNA expression profiling, has positioned miRNAs as biomarker candidates.

Altered miRNA expression has been reported in several human autoimmune diseases, and miR-92a was suggested as a circulating biomarker for disease staging in MS. The single study on miRNAs in NMO revealed that several miRNAs have distinct expression levels in NMO patients compared with healthy controls (HCs) and patients with MS [9].

For many years, NMO was considered as a severe variant of MS [1]. Both diseases have autoimmune inflammatory lesions in the CNS. In MS there is an abundance of data on miRNA expression in whole blood, peripheral blood mononuclear cells (PBMCs), plasma, cerebrospinal fluid (CSF), and peripheral blood T cells [31-34]. miRNAs are dysregulated in MS, but their use as biomarkers is still evolving [31]. In a publication reviewing miRNAs in MS, Xinting et al. [44] summarized that miR-15a, miR-19a, miR-22, miR-210 and miR-223 were up- regulated in T-reg cells, plasma, blood cells, PBMCs and brain white matter tissues from MS patients; miR-21, miR-142-3p, miR-146a, miR-146b, miR-155 and miR- 326 were up-regulated and miR-181c and miR-328 were down-regulated in PBMCs and brain lesions; and miR-15a and miR-15b were down-regulated in blood, peripheral T cells and B cells or plasma samples from MS patients.

In MS, there have been limited attempts to study the use of miRNAs in treated versus untreated patients, evaluating commonly used therapies. For example, Waschbisch et al. studied five miRNAs (miR-20b, miR-142-3p, miR-146a, miR-155, and miR-326) by qPCR and found that there was significantly lower expression of miR-142-3p and miR-146a in glatiramer acetate (GA) treated patients [35]. In an investigation of miRNA and mRNA expression in PBMCs of MS patients before and after IFN-β therapy, Hecker et al. found that IFN-β -responsive genes were upregulated in parallel to down-regulation of miRNAs. Among the miRNAs, they identified altered expression among members of the mir-29 family [36]. Given the low clinical response rate of MS patients to IFN-β and GA therapy, the implications of these studies are unclear.

For a more effective MS therapy (natalizumab), Sievers et al. found that 10 miRNAs, out of 1059 tested, were differentially expressed in B cells of natalizumab- treated patients versus untreated patients and Ingwersen et al found that natalizumab therapy restored aberrant blood miRNA expression profiles in MS patients [37, 38, respectively]. The 10 most strongly upregulated miRNAs from MS lesions were expressed in astrocytes.

The most commonly currently used treatments of NMO are steroids, azathioprine, mycophenolate mofetil, and rituximab [10-13]. Rituximab is a chimeric anti-CD20 monoclonal antibody that depletes B cells. It is commonly used for treating B cell lymphoma and has been found to be effective in the treatment of autoimmune rheumatological and neurological conditions, including NMO. Rituximab is currently considered the most effective therapy for preventing NMO exacerbations [14, 15]. Data relating to the correlation between AQP4-IgG titers and disease activity in the long-term course of NMO are inconsistent [16]. Previous studies testing rituximab in NMO found a high response rate and most of the patients remained relapse free or experienced decreases in the annualized relapse rate (ARR) (60-100% of disease-free patients in cohorts with 5-30 NMO patients) [22, 23].

Currently, no definite biomarker of response to therapy is available for monitoring patients with NMO. Further, it is challenging to diagnose NMO patients that are negative for the AQP4-IgG marker from patients with other CNS demyelinating diseases, especially multiple sclerosis (MS). Several attempts have been made to develop biomarkers in order to individualize rituximab therapy in NMO patients. CD19+ and CD27+CD19+B cell counts were studied by Kim et al in NMO patients treated with rituximab [22, 42]. Recently it was found that, NMO patients carrying the V158F allele of the fragment c gamma receptor 3 A (FCGR3A- F) have increased likelihood of incomplete B-cell depletion and 5-fold increase chance of relapse following rituximab treatment [43].

Thus, there is a need in the art for biomarkers and corresponding methods that can be reliably used for monitoring and efficiently determining treatment response in NMO patients and/or MS patients. Such biomarkers should preferably be found in peripheral blood of patients and can be used as predictor for response to therapy, for assisting in the individualized management of the disease. SUMMARY OF THE INVENTION

According to some embodiments, there are provided methods for determining response to therapy in a subject afflicted with Neuromyelitis optica (NMO). In some embodiments, the methods include identification of miRNA biomarkers in the peripheral blood of patients with NMO.

According to some embodiments, there are provided methods for determining response to therapy in a subject afflicted with Multiple Sclerosis (MS). In some embodiments, the method include identification of miRNA biomarkers in the peripheral blood of patients with MS.

In some embodiments, the therapy includes administration of a suitable treatment. In some embodiments, the treatment may be selected from: Rituximab, B cell depletion treatment, treatment with anti CD20, treatment with anti-CD19, ocrelizumab, ofatumumab (HuMax-CD20), Natalizumab, azathioprine, Cladribine, Lemtrada, Teriflunomide, and the like, or combinations thereof.

In some embodiments, analysis of global miRNA expression in the blood of patients with NMO before and after suitable treatment (such as, for example, rituximab) identified a distinct miRNA expression signature (profile) associated with the treatment.

In some embodiments, analysis of global miRNA expression in the blood of patients with MS before and after treatment (such as, rituximab) results in a distinct miRNA expression signature associated with the treatment.

In some embodiments, a set of miRNAs with elevated expression in the blood of MS patients revert to the levels of matched healthy controls following effective therapy (for example, with rituximab).

In some embodiments, a set of miRNAs with elevated expression in the blood of NMO patients revert to the levels of matched healthy controls following effective therapy (for example, with rituximab). In some embodiments, at least some of the miRNAs are brain-enriched miRNAs.

According to some embodiments, there is provided a method for determining efficacy of treatment for neuromyelitis optica (NMO) in a subject in need thereof, the method comprising:

a) determining the levels of at least two circulating miRNAs, selected from the group consisting of miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180, in a first serum sample of said subject; b) treating said subject with a selected treatment;

c) determining the levels of the at least two circulating miRNAs in a second serum sample of said subject; and

d) comparing the levels of the at least two circulating miRNAs in said first and second serum samples,

wherein modulation in the levels of the at least circulating miRNAs from the first to the second serum sample is indicative of said treatment efficacy.

According to some embodiments, step (a) may include determining the levels of at least three circulating miRNAs, selected from the group consisting of miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180.

According to some embodiments, the selected treatment may be selected from, but not limited to: Rituximab, B cell depletion treatment, treatment with anti CD20, treatment with anti-CD 19, ocrelizumab, ofatumumab (HuMax-CD20), Natalizumab, azathioprine, Cladribine, Lemtrada, and Teriflunomide.

In some embodiments, the selected treatment includes Rituximab.

In some embodiments, step a) may be conducted at a time point prior to step b).

In some embodiments, steps a) and c) may be conducted at distinct time points during step b). In some embodiments, the method may further include repeating step c) for one or more times to determine the levels of the at least two circulating miRNAs in consecutive serum samples, obtained at designated time intervals; and comparing the levels of the at least two circulating miRNAs between said consecutive serum samples, wherein a modulation in the levels of the at least two circulating miRNAs between consecutive serum samples is indicative of said treatment efficacy.

In some embodiments, modulation comprises decrease in the level of the at least two miRNAs. In some embodiments, modulation comprises an increase.

In some embodiments, the method may further include a step of isolating RNA from the first serum sample and/or the second serum prior to identification of the at least two miRNAs.

In some embodiments, the level of the at least two miRNAs in the first serum sample and/or the second serum sample may be determined by a method selected from: amplification reaction, sequencing reaction, microarray, or combinations thereof.

In some embodiments, the treatment regime with the selected treatment is adjusted based on the determined treatment efficacy.

In some embodiments, the method may further include a step of comparing the levels of the at least two circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of corresponding miRNAs obtained from healthy control subjects.

According to some embodiments there is provided a method for assessing treatment efficacy of a selected treatment in a subject afflicted with neuromyelitis optica (NMO), the method comprising determining the level of at least two circulating miRNA molecules, selected from the group consisting of: miR-138, miR- 660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180, in serum samples of said subjects, wherein said samples are obtained before and after treatment with the selected treatment and comparing the levels of the at least two circulating miRNA molecules between the serum samples, wherein modulation of expression of the miRNA molecules between the serum samples obtained before and after treatment is indicative of increased treatment efficacy.

According to some embodiments, the methods for determining treatment efficacy of NMO patient, include evaluating the treatment efficacy by clinical parameters, including determining the level of anti Aquaporin 4 expression levels.

According to some embodiments, there is provided a kit for determining efficacy of a selected treatment for neuromyelitis optica (NMO), the kit includes means for determining the levels of at least two circulating miRNAs, selected from: miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180 in serum samples of a subject, said samples obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for the neuromyelitis optica (NMO).

In some embodiments, the means in the kit may include specific nucleic acid molecules for identification of circulating miRNAs in the serum samples. In some embodiments, the nucleic acid molecules may include specific primers for identification of miRNAs in an amplification reaction performed on RNA isolated from the serum sample. According to some embodiments, the nucleic acid molecules include specific probes for identification of circulating miRNAs in a sequencing reaction performed on RNA or DNA isolated from the serum sample.

In some embodiments, the kit instructions further include comparing the levels of circulating miRNAs in the serum samples of said subject to the circulating levels of miRNAs obtained from healthy control subjects.

According to some embodiments, there is provided method for determining treatment efficacy of multiple sclerosis (MS) in a subject in need thereof, said method comprising:

a) determining the levels of at least two circulating miRNAs, selected from the group consisting of miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180, in a first serum sample of said subject; b) treating said subject with a selected treatment; c) determining the levels of the at least two circulating miRNAs in a second serum sample of said subject; and

According to some embodiments, the selected treatment is selected from: Rituximab, B cell depletion treatment, treatment with anti CD 20, treatment with anti- CD 19, ocrelizumab, ofatumumab (HuMax-CD20), Natalizumab, azathioprine, Cladribine, Lemtrada, and Terifiunomide.

In some embodiments, step a) may be conducted at a time point prior to step b).

In some embodiments, steps a) and c) may be conducted at distinct time points during step b).

According to some embodiments, the method may further include repeating step c) for one or more times to determine the levels of the at least two circulating miRNAs in consecutive serum samples, obtained at designated time intervals; and comparing the levels of the at least two circulating miRNAs between said consecutive serum samples, wherein a modulation in the levels of the at least two circulating miRNAs between consecutive serum samples is indicative of said treatment efficacy.

In some embodiments, the modulation may include decrease or increase in the level of the at least two miRNAs.

According to some embodiments, the treatment regime using the selected treatment may be adjusted based on the determined treatment efficacy.

In some embodiments, the method may further include a step of comparing the levels of the at least two circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of corresponding miRNAs obtained from healthy control subjects. According to some embodiments, there is provided a method for assessing treatment efficacy of a selected treatment in a subject afflicted with Multiple Sclerosis (MS), the method comprising determining the level of at least two circulating miRNA molecules, selected from the group consisting of: miR-138, miR- 660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180, in serum samples of said subjects obtained before and after treatment with the selected treatment, and comparing the levels of the at least two circulating miRNA molecules between the serum samples, wherein modulation of expression of the miRNA molecules between the serum samples obtained before and after treatment is indicative of increased treatment efficacy.

In some embodiments the method may further include comparing the at least two circulating miRNA molecules between the consecutive serum samples.

In some embodiments, the method may further include evaluating the treatment efficacy by clinical parameters.

In some embodiments, the modulation includes increased levels of the miRNAs or reduced levels of the miRNAs.

In some embodiments, the method may further include a step of comparing the levels of circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of miRNAs obtained from healthy control subjects.

According to some embodiments, there is provided a kit for determining efficacy of a selected treatment for multiple sclerosis (MS), the kit includes means for determining the levels of at least two circulating miRNAs, selected from: miR- 138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR- 203a, miR-124, miR-7 and miR-1180 in serum samples of a subject, said samples obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for MS.

BRIEF DESCRIPTION OF THE FIGURES

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

Fig. 1- Graphs showing differentially expressed miRNAs in circulating blood of NMO patients (responders and non-responders) patients 6 month following Rituximab therapy. RNA was extracted from whole blood of 9 responders and 5 non- responders NMO patients 6 month following Rituximab therapy and miRNAs were quantified using Real time RT PCR. The results of 3 most significant differentially expressed miRNAs are presented in the figure as 2^ΛΔ ct. Responders are marked in dark black and non-responders in grey.

Figs. 2A-B: Graphs showing differential expression of the indicated miRNAs between Healthy Controls and NMO patients before and following Rituximab therapy. RNA was extracted from whole blood of 15 healthy controls and 9 NMO patients before and after Rituximab therapy and miRNAs were quantified using deep sequencing. The results of 9 of the 10 miRNAs which revert to normal levels following Rituximab therapy are presented in the figure as normalized counts (logarithmic scale). Healthy controls, NMO patients before Rituximab therapy and NMO patients after therapy are marked as circles, squares and triangles, respectively.

DETAILED DESCRIPTION OF THE INVENTION

According to some embodiments, the present invention provides methods and kits for determining treatment efficacy of suitable drug in a patient afflicted with Neuromyelitis Optica (NMO) or Multiple Sclerosis (MS), wherein circulating levels of designated miRNAs are used as biomarkers.

The present invention discloses for the first time the unexpected discovery that the detection of circulating miRNA in serum of NMO or MS patients treated with a suitable drug (such as, for example, rituximab) reflects the treatment efficacy of the treated subject. The methods currently used for assessing treatment efficacy are complicated and cumbersome and in many cases provides false or non-accurate results. Hence, the use of detecting circulating levels of specific miRNAs, (in particular, one or more of: miR-138, miR-660, miR-125b, miR-760, miR-100, miR- 423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180) as a biomarker of treatment efficacy of suitable treatment, overcomes the drawbacks of currently used methods in providing a more sensitive, reliable, simple and cost effective method for determine efficacy of such treatment.

Thus, in some embodiments, the present invention provides methods, kits and compositions for assessing or determining the effectiveness of an treatment of NMO or MS patients, based on the modulation in the circulating levels of miRNAs, in particular, one or more miRNA's selected from: miR-138, miR-660, miR-125b, miR- 760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7, miR- 1180 and any combination thereof. Each possibility is a separate embodiment.

To facilitate an understanding of the present invention, a number of terms and phrases are defined below. It is to be understood that these terms and phrases are for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

As referred to herein, the terms "polynucleotide molecules", "oligonucleotide", "polynucleotide", "nucleic acid" and "nucleotide" sequences may interchangeably be used. The terms are directed to polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA), and modified forms thereof in the form of a separate fragment or as a component of a larger construct, linear or branched, single stranded (ss), double stranded (ds), triple stranded (ts), or hybrids thereof. The term also encompasses RNA/DNA hybrids. The polynucleotides may be, for example, sense and antisense oligonucleotide or polynucleotide sequences of DNA or RNA. The DNA or RNA molecules may be, for example, but are not limited to: complementary DNA (cDNA), genomic DNA, synthesized DNA, recombinant DNA, or a hybrid thereof or an RNA molecule such as, for example, mRNA, shRNA, siRNA, miRNA, and the like. Accordingly, as used herein, the terms "polynucleotide molecules", "oligonucleotide", "polynucleotide", "nucleic acid" and "nucleotide" sequences are meant to refer to both DNA and RNA molecules. The terms further include oligonucleotides composed of naturally occurring bases, sugars, and covalent inter nucleoside linkages, as well as oligonucleotides having non-naturally occurring portions, which function similarly to respective naturally occurring portions.

As referred to herein, the term "Treating a disease" or "treating a condition" is directed to administering a composition, which comprises at least one reagent (which may be, for example, one or more polynucleotide molecules, one or more expression vectors, one or more substance/ingredient, and the like), effective to ameliorate symptoms associated with a disease, to lessen the severity or cure the disease, or to prevent the disease from occurring.

As used herein the terms "diagnosing" or "diagnosis" refer to the process of identifying/detecting/assessing NMO and/or MS in a subject.

The term "organism" refers to a mammal. In some embodiments, the organism is human. In some embodiments, the organism is selected from a pet, a rodent, a farm animal, and a lab animal.

As used herein, the term "subject" is interchangeable with an individual or patient. According to some embodiments, the subject is a mammal. According to some embodiments, the subject is a human. According to some embodiments, the subject is symptomatic. According to other embodiments, the subject is asymptomatic. According to some embodiments, the subject may be treated with one or more drugs or other suitable treatment.

As used herein, the term "small interfering RNA" and "siRNA" are used interchangeably and refer to a nucleic acid molecule mediating RNA interference or gene silencing. The siRNA inhibits expression of a target gene and provides effective gene knock-down. The terms "microRNA" and "miRNA" are directed to a small non-coding RNA molecule that can function in transcriptional and post-transcriptional regulation of target gene expression.

The term "circulating" with respect to miRNA molecules is directed to the presence of the miRNA in circulating blood (serum) and not localized to an organ or localized to a specific organ (such as the brain).

As used herein the term "biological sample" refers to any sample obtained from the subject being tested. According to some embodiments, the biological sample is a biopsy. According to some embodiments, the biological sample is selected from: cells, tissue and bodily fluid. Each possibility represents a separate embodiment of the invention. According to some embodiments, the biological sample is a fluid sample. According to some embodiments, the fluid sample is selected from the group consisting of: whole blood, plasma, serum, and the like. Each possibility represents a separate embodiment of the invention. In some embodiments, the biological sample is obtained or collected from the subject in any method known in the art. The sample may be collected from the subject by noninvasive, invasive or minimal invasive means. Each possibility represents a separate embodiment of the invention. According to some embodiments, the sample may be treated prior to being subjected to the methods of the present invention. According to some embodiments, the sample is a fluid sample and is substantially free of residual cells or debris of cells. For removing cells or debris of cells from within a bodily fluid sample, said cells or debris of cells may be precipitated by centrifugation and the supernatant is taken for determining the specific biomarkers levels. Typically, centrifugation of up to 2500 revolutions per minute (rpm) for up to 20 min is performed. A common centrifugation procedure is associated with centrifugation of 1100 rpm for 7 min, or centrifugation of 2000 rpm for 5 min. Alternatively, cells can be removed by filtration. According to some embodiments, the liquid sample undergoes concentration with a suitable membrane pore cut-off size. In accordance with some embodiments, a fluid sample treated with such filter is concentrated, namely, any molecule, particle below the size of the cut-off of the filter is removed. According to some embodiments, the liquid sample is concentrated by up to 100 times the initial concentration. According to some embodiments, the sample is a fluid sample, such as, serum, and the miRNA molecules within said fluid sample are analysed. In accordance with some embodiments, the sample is collected, centrifuged, the supernatant is removed and analysed for specific miRNA molecules. According to some embodiments, the sample is reconstituted (e.g. with fluids, such as, PBS or media). According to alternative embodiments the fluid sample is analyzed together with the cells present therein without prior separation. According to additional embodiments, the sample may conveniently be frozen after being collected from the subject and thawed before use. In some embodiments, the biological sample may also optionally comprise a sample that has not been physically removed from the subject.

According to some embodiments, the methods of the invention encompass determining the circulating levels of miRNAs in serum samples of a subject afflicted with NMO or MS, wherein the samples are obtained before and after suitable treatment. In some embodiments, the serum samples may be obtained at various time points before commencement of treatment. In some embodiments, the time points before commencement of treatment may be any time point of between 1 hour to 6 months. In some embodiments, the time points before commencement of treatment may be any time point of between 1-2 months. In some embodiments, the serum samples may be obtained at various time points after commencement of treatment. In some embodiments, the time points after commencement of treatment may be any time point of between 1 hour to 24 months. In some embodiments, the time points after commencement of treatment may be any time point of between 1 month to 24 months. In some embodiments, the time points after commencement of treatment may be any time point of between 2 to 12 weeks. In some embodiments, the time points after commencement of treatment may be any time point of between 1 hour to 12 months. In some embodiments, the intervals between time points obtaining of serum samples may be predetermined. In some embodiments, the intervals between time points of obtaining of serum samples may be identical or different. For example, the intervals may include intervals of 1 day, one week, one month (i.e., one month between consecutive samples). For example, the intervals may include intervals of 2-12 weeks. For example, the intervals may include intervals of 1-4 weeks. For example, the intervals may include intervals of 4-10 weeks. In some embodiments, the number of samples obtained before and/or after treatment as well as the time intervals between obtaining the samples may be predetermined or may be determined based on the patient condition, treatment regime, and the like.

According to some embodiments, the method of assessing treatment efficacy of NMO or MS patients may encompass, apart for the determining the levels of the indicated circulating miRNA's, any known methodologies used to assess the respective condition and/or treatment efficacy.

As used herein the term "elevation" or "increase" of circulating levels of miRNAs refers, according to some embodiments, to a statistically significant elevation. As used herein the term "decrease" or "reduction" of circulating levels of miRNAs refers, according to some embodiments, to a statistically significant decline. According to some embodiments, level is amount. According to some embodiments, levels of miRNAs reflect the expression, secretion and/or presence of these nucleotide molecules in a serum sample of a subject. According to some embodiments, levels of miRNAs in the serum reflect the expression/presence/secretion of these polynucleotides from within cells or tissues, such as, brain tissue.

As used herein the term "modulation" of circulating levels of miRNAs refers to the change in the levels. In some embodiments, modulation is increase in the levels. In some embodiments, modulation is a decrease in the levels. In some embodiments, the changes may be between various measurements obtained at different time points. In some embodiments, the changes may be between consecutive measurements. In some embodiments, the changes may be between various measurements obtained before and after treatment.

According to some embodiments, the results presented herein demonstrate for the first time that a set of miRNAs with elevated expression in the blood of NMO patients revert to the levels of matched healthy controls following effective therapy with rituximab. The majority of these "normalized" miRNAs are known as brain specific/enriched miRNAs. Thus, a diagnostic test to monitor treatment response in NMO is provided herein. According to some embodiments, the results presented herein demonstrate that specific miRNA signatures in whole blood of patients with NMO can serve as biomarkers for therapy response. Furthermore, monitoring the levels of brain specific/enriched miRNAs in the blood can reflect the degree of disease activity in the CNS of inflammatory demyelinating disorders.

According to some embodiments, the brain enriched miRNAs disclosed herein, that revert to healthy control levels after treatment are expressed abundantly or predominantly in the brain. Without wishing to be bound to any theory or mechanism, the higher quantity of these miRNAs in the circulation of patients with NMO may imply that they were shed to the circulation by degenerative brain cells, with a corresponding decrease in the amount of these miRNAs in the circulation observed following treatment, when the destructive inflammatory process diminishes. Therefore, monitoring the levels of brain specific/enriched miRNAs in the blood reflects the degree of disease activity in the CNS of demyelinating disorders.

According to some embodiments, there is provided a method for determining response to therapy in a subject afflicted with Neuromyelitis optica (NMO). In some embodiments, the methods include identification of miRNA biomarkers in the peripheral blood of patients with NMO.

According to some embodiments, there is provided a method for determining efficacy of treatment for neuromyelitis optica (NMO) in a subject in need thereof, the method comprising one or more of the steps of:

a) determining the levels of one or more circulating miRNAs, in a first serum sample of said subject;

b) treating said subject with a selected treatment;

c) determining the levels of the circulating miRNAs in a second serum sample of said subject; and

d) comparing the levels of the circulating miRNAs in said first and second serum samples,

wherein modulation in the levels of the circulating miRNAs from the first to the second serum sample is indicative of said treatment efficacy. In some embodiments, step (a) may include determining the levels of at least two circulating miRNAs. In some embodiments, step (a) may include determining the levels of at least three circulating miRNAs.

In some embodiments, step a) may be conducted at a time point prior to step b).

In some embodiments, the method may further include repeating step c) for one or more times to determine the levels of the circulating miRNAs in consecutive serum samples, obtained at designated time intervals; and comparing the levels of the circulating miRNAs between said consecutive serum samples, wherein a modulation in the levels of the at circulating miRNAs between consecutive serum samples is indicative of said treatment efficacy.

In some embodiments, the method may further include a step of isolating RNA from the first serum sample and/or the second serum prior to identification of the at miRNAs.

In some embodiments, the method may further include a step of comparing the levels of the circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of corresponding miRNAs obtained from healthy control subjects.

According to some embodiments, there is provided a method for determining response to therapy in a subject afflicted with Multiple Sclerosis (MS). In some embodiments, the method include identification of miRNA biomarkers in the peripheral blood of patients with MS.

According to some embodiments, there is provided a method for determining efficacy of treatment for multiple sclerosis (MS) in a subject in need thereof, the method comprising one or more of the steps of:

b) treating said subject with a selected treatment; c) determining the levels of the circulating miRNAs in a second serum sample of said subject; and

wherein modulation in the levels of the circulating miRNAs from the first to the second serum sample is indicative of said treatment efficacy.

In some embodiments, step (a) may include determining the levels of at least two circulating miRNAs. In some embodiments, step (a) may include determining the levels of at least three circulating miRNAs.

In some embodiments, step a) may be conducted at a time point prior to step b).

In some embodiments, the method may further include a step of isolating

RNA from the first serum sample and/or the second serum prior to identification of the at miRNAs.

In some embodiments, a set of miRNAs with elevated expression in the blood of NMO patients revert to the levels of matched healthy controls following effective therapy (for example, with rituximab).

In some embodiments, the miRNAs are brain enriched miRNAs.

In some embodiments, the brain enriched miRNAs may be selected from: miR-135a, miR-135b, miR-125b, miR-134, miR-138 and/or miR-76.

In some embodiments, the methods and kits disclosed herein include determining the levels of one or more circulating miRNAs selected from: miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180. Each possibility is a separate embodiment.

In some embodiments, the methods and kits disclosed herein include determining the levels of one or more circulating miRNAs selected from: miR-138, miR-660, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124 and miR-7. Each possibility is a separate embodiment.

According to some embodiments, the methods and kits disclosed herein include determining the levels of at least two miRNAs, selected from the group consisting of: miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR- 135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180. Each possibility is a separate embodiment.

According to some embodiments, the methods and kits disclosed herein include determining the levels of at least three miRNAs, selected from the group consisting of: miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR- 135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180. Each possibility is a separate embodiment.

According to some embodiments, the methods and kits disclosed herein include determining the levels of miR-1180 and miR-125b and at least one additional miR selected from the group consisting of: miR-203, miR-138, miR-660, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124 and miR-7. Each possibility is a separate embodiment.

In some embodiments, the circulating miRNAs may be selected from one or more of the miRNAs listed in Table 1A and/or Table IB and/or Fig. 1 and/or Figs. 2A-2B.

According to some embodiments, as disclosed herein, a miRNA signature in whole blood of patients with NMO was identified. Following rituximab therapy, the expression levels of the majority of miRNAs that have higher expression in patients with NMO versus healthy controls (HCs) revert to normal levels following therapy (such as, rituximab). In some embodiments, this miRNA signature can be used as a biomarker for therapy response. In some embodiments, at least some of the identified miRNAs are brain-enriched miRNAs. In some embodiments, monitoring the levels of brain specific miRNAs in the blood of patients with inflammatory demyelinating disorders can reflect the degree of disease activity in the CNS.

In some embodiments, the methods disclosed herein relates to specific miRNA signatures in whole blood of patients with MS that can serve as biomarkers for therapy response. Furthermore, monitoring the levels of brain specific/enriched miRNAs in the blood can reflect the degree of disease activity in the CNS of inflammatory demyelinating disorders.

In some embodiments, there is provided a method for determining efficacy of treatment for neuromyelitis optica (NMO) in a subject in need thereof, said method comprising: determining the levels of circulating miRNAs in a first serum sample of said subject; treating said subject with a selected treatment; determining the levels of circulating miRNAs in a second serum sample of said subject; and comparing the levels of the circulating miRNAs in said first and second serum samples, wherein modulation in the levels of circulating miRNAs from the first to the second serum sample is indicative of said treatment efficacy.

In some embodiments, the method further comprising a step of isolating RNA from the first serum sample and/or the second serum prior to identification of the miRNA. In some embodiments, the level of miRNA in the first serum sample and/or the second serum sample may be determined by a method selected from: amplification reaction, sequencing reaction, microarray, or combinations thereof.

In some embodiments, the treatment regime using the selected treatment is adjusted based on the determined treatment efficacy.

In some embodiments, the method further includes a step of comparing the levels of circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of miRNAs obtained from healthy control subjects.

In some embodiments, modulating comprises increase in the level of miRNA and/or decrease in the level of miRNA. In some exemplary embodiments, modulating includes increase in the level of miRNA.

In some embodiments, there is provided a method for assessing treatment efficacy of a selected treatment in a subject afflicted with neuromyelitis optica (NMO), the method comprising determining the level of circulating miRNA molecules in serum samples of said subjects obtained before and after treatment with the selected treatment, and comparing the levels of the circulating miRNA molecules between the serum samples, wherein modulation of expression of the miRNA molecules between the serum samples obtained before and after treatment is indicative of increased treatment efficacy.

In some embodiments, there is provided a kit for determining efficacy of a selected treatment for neuromyelitis optica (NMO), comprising means for determining the levels of circulating miRNAs in serum samples of a subject obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for the neuromyelitis optica (NMO).

According to some embodiments, there is provided a kit for determining efficacy of a selected treatment for NMO, the kit includes means for determining the levels of one or more circulating miRNAs, selected from: miR-138, miR-660, miR- 125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180 in serum samples of a subject, said samples obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for NMO.

In some embodiments, there is provided a method of predicting responsiveness of a subject afflicted with neuromyelitis optica (NMO) to Rituximab treatment, the method comprising determining the level of a plurality of circulating miRNAs, comprising one or more of the miRNAs listed in Table IB, in a serum sample obtained from the subject, wherein a significant difference between the level of said circulating miRNAs in said sample, compared to a control value is indicative of the responsiveness of said subject to Rituximab treatment.

In some embodiments, there is provided a method for predicting responsiveness treatment of a subject afflicted with neuromyelitis optica (NMO), said method comprising: determining the levels of circulating miRNAs in a first serum sample of said subject and comparing the levels of the circulating miRNAs in said first serum sample to a control value; wherein a significant difference between the level of said circulating miRNAs in said sample, compared to a control value is indicative of the responsiveness of said subject to said treatment.

In some embodiments, there is provided a method for determining efficacy of treatment for MS in a subject in need thereof, said method comprising: determining the levels of circulating miRNAs in a first serum sample of said subject; treating said subject with a selected treatment; determining the levels of circulating miRNAs in a second serum sample of said subject; and comparing the levels of the circulating miRNAs in said first and second serum samples, wherein modulation in the levels of circulating miRNAs from the first to the second serum sample is indicative of said treatment efficacy. In some embodiments, there is provided a method for assessing treatment efficacy of a selected treatment in a subject afflicted with MS, the method comprising determining the level of circulating miRNA molecules in serum samples of said subjects obtained before and after treatment with the selected treatment, and comparing the levels of the circulating miRNA molecules between the serum samples, wherein modulation of expression of the miRNA molecules between the serum samples obtained before and after treatment is indicative of increased treatment efficacy.

In some embodiments, there is provided a kit for determining efficacy of a selected treatment for MS, comprising means for determining the levels of circulating miRNAs in serum samples of a subject obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for the MS.

According to some embodiments, there is provided a kit for determining efficacy of a selected treatment for multiple sclerosis (MS), the kit includes means for determining the levels of one or more circulating miRNAs, selected from: miR- 138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR- 203a, miR-124, miR-7 and miR-1180 in serum samples of a subject, said samples obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for MS.

In some embodiments, there is provided a method of predicting responsiveness of a subject afflicted with MS to Rituximab treatment, the method comprising determining the level of a plurality of circulating miRNAs, comprising one or more of the miRNAs listed in Tables 1 A and/or Tables IB, in a serum sample obtained from the subject, wherein a significant difference between the level of said circulating miRNAs in said sample, compared to a control value is indicative of the responsiveness of said subject to Rituximab treatment.

In some embodiments, there is provided a method for predicting responsiveness treatment of a subject afflicted with MS, said method comprising: determining the levels of circulating miRNAs in a first serum sample of said subject and comparing the levels of the circulating miRNAs in said first serum sample to a control value; wherein a significant difference between the level of said circulating miRNAs in said sample, compared to a control value is indicative of the responsiveness of said subject to said treatment.

According to some embodiments, one or more first samples may be taken at a time point prior to initiation of the treatment and one or more second samples are taken at a time point during or after the treatment. According to some embodiments, one or more first samples may be taken at a time point during the treatment (step b) and one or more second samples may be taken at a time point during the treatment and subsequent to the time point of the one or more first samples. According to some embodiments, one or more first samples are taken at a time point during the treatment (step b) and one or more second samples are taken at a time point after the treatment has been discontinued. According to some embodiments, one or more second samples may be obtained consecutively, at designated time intervals (such as, in the range of 1 -10 weeks). According to some embodiments, a decrease in the level of the miRNAs in the at least one second sample as compared to that determined for the first sample or compared to a previous consecutive second serum sample, is indicative that the treatment is effective.

In some embodiments, the control value is zero (i.e., non detectable level). In some embodiments, the control value is any predetermined value. In some embodiments, the methods are qualitative. In some embodiments, the methods are quantitative.

In some embodiments, identification and/or quantification of the specific miRNAs in the serum may be performed by use of specific probes or tags capable of specifically interacting with the miRNA molecules. In some embodiments, such tags or probes may include nucleic acid molecules. In some embodiments, such tags or probes may include nucleic acid probe, nucleic acid primers, peptidic-tags, and the like.

According to some embodiments, suitable methods to detect and/or quantify miRNA may include such methods as, but not limited to: amplification reaction, such as, PCR, RT-PCR, real time PCR, and the like; Gene expression microarrays, such as Affymetrix, Agilent, and Illumina microarray platforms; NanoString technology, such as nCounter technology which employs unique fluorescent-tagging of individual miRNA species followed by two-dimensional display and optical scanning and counting of miRNA molecules; Sequencing, such as, next Generation Sequencing (NGS) using specific adaptors and/or probes; and the like, or combinations thereof.

In some embodiments, the level of the circulating miRNAs may be determined at one or more time points after initiation of treatment. In some embodiments, the level of the circulating miRNAs may be determined at various time points after initiation of treatment. In some embodiments, the levels of circulating miRNAs may be compared between consecutive measurements (i.e., measurements obtained between two samples obtained at consecutive time points) and treatment efficacy is determined based on the comparison.

In some embodiments, the method may further include evaluating the disease status by clinical parameters. In some embodiments, there is a correlation between circulating levels of the miRNAs and the clinical parameters.

In some embodiments, the methods of the invention are useful for managing the subject treatment by the clinician or physician subsequent to the determination of treatment efficacy. According to some embodiments, based on the determination of the treatment efficacy, treatment regime may be adjusted or modulated. Adjustment of treatment regime may include, for example, but not limited to: adjustment of dosage (reducing or increasing dosage), ceasing treatment, replacing or adding a drug, and the like.

According to some embodiments, the results presented herein demonstrate for the first time that a set of miRNAs with elevated expression in the blood of NMO patients revert to the levels of matched healthy controls following effective therapy with rituximab. The majority of these "normalized" miRNAs are known as brain specific/enriched miRNAs. Thus, a diagnostic test to monitor treatment response in NMO is provided herein.

According to some embodiments, the results presented herein demonstrate that specific miRNA signatures in whole blood of patients with NMO can serve as biomarkers for therapy response. Furthermore, monitoring the levels of brain specific/enriched miRNAs in the blood can reflect the degree of disease activity in the CNS of inflammatory demyelinating disorders. According to some embodiments, the brain enriched miRNAs disclosed herein, that revert to healthy control levels after treatment are expressed abundantly or predominantly in the brain. Without wishing to be bound to any theory or mechanism, the higher quantity of these miRNAs in the circulation of patients with NMO may imply that they were shed to the circulation by degenerative brain cells, with a corresponding decrease in the amount of these miRNAs in the circulation observed following treatment, when the destructive inflammatory process diminishes. Therefore, monitoring the levels of brain specific/enriched miRNAs in the blood reflects the degree of disease activity in the CNS of demyelinating disorders. In some embodiments, without wishing to be bound to any theory or mechanism, in NMO subjects, the autoantibodies that are directed against aquaporin 4 (AQP4) are expressed by astrocytes. This may indicate that miRNAs may contribute to pathogenesis of diseases in which astrocytes play a major role, like NMO.

According to some embodiments, as presented herein, there are differentially expressed miRNAs in NMO patients and HCs, as well as differential miRNA expression after treatment. Accordingly, a signature of miRNA expression was identified that not only differed between NMO patients and HCs, but also reverted to normal in response to therapy (such as, rituximab therapy).

In some embodiments, without wishing to be bound to any theory or mechanism, as presented in the examples herein, the blood samples taken at the six- month time point were taken following analysis of positive CD 19 cell detection in the blood, indicating that patients were not depleted of B cells. This supports the notion that the miRNAs signature represents a distinct phenomenon, rather than simply being a reflection of decreased B cell counts. The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". The terms "comprises" and "comprising" are limited in some embodiments to "consists" and "consisting", respectively. The term "consisting of" means "including and limited to". The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure. As used herein the term "about" in reference to a numerical value stated herein is to be understood as the stated value +/- 10%.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

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.

The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Materials and Methods

Subjects:

NMQ: The patient cohort included 16 patients with NMO (14 females, 2 males; age 41+14.3 years; disease duration, 5.3+6.2 years; EDSS [Expanded Disability Status Scale], 4.8+1.8), followed at the Hadassah MS Center. The relapse rate in the 2 years prior to the study was 1.25+0.7. None of the enrolled patients had relapse or were treated with steroids for at least 30 days before their blood samples were drawn. 82.3% of the NMO patients were positive for anti AQP4. Brain magnetic resonance imaging (MRI) was normal or compatible with the diagnosis of NMO in all patients, and spinal MRIs revealed long extensive myelitis in 14/16 patients. None of the 9 responders had relapses in the 2 years following rituximab therapy. 5 patients with treatment failure had comparable clinical characteristics to the responders but experienced no decrease in relapse rate with rituximab therapy (4 females, 1 male; age 46+10.7 years; disease duration, 5.2+4.0 years; EDSS 5.2+4.4, relapse rate in the 2 years prior to the study, 1.3+0.44). The patients signed informed consent. Clinical data were collected from patients' files. NMO patients were diagnosed according to the NMO diagnostic criteria [17]. An age- and sex-matched control group included 15 healthy individuals (10 females, 5 males; age 36.7+9.4 years).

MS: The patient cohort includes additional 20 patients with MS that respond to rituximab/ocrelizumab therapy, 20 patients with MS that did not respond to the rituximab/ocrelizumab therapy followed at the Hadassah MS Center. Clinical data collected from patients' files. MS patients are diagnosed according to the diagnostic criteria. An age- and sex- matched control group included 15 healthy individuals.

The patient cohort includes additional 20 patients with MS that respond to TYSABRI® (natalizumab) therapy, 20 patients with MS that did not respond to the TYSABRI® (natalizumab) therapy, followed at the Hadassah MS Center. Clinical data is collected from patients' files. MS patients are diagnosed according to the MacDonald's diagnostic criteria. An age- and sex- matched control group include 15 healthy individuals. NMO-Ig seropositivity

Cohort was determined using RSR ELISA assay in sera. The anti-AQP4 ELISA positive samples were also assessed using a cell-based assay (Euroimmun).

Blood RNA isolation and miRNA quantification

A 4-ml blood sample was collected in EDTA tubes from each patient. 250 μΐ of whole blood was mixed with 750 μΐ of Tri-reagent BD (Sigma), supplemented with 20 μΐ of 5N acetic acid, and frozen. RNA was extracted according to the manufacturer's instructions. Gel Electrophoresis confirmed the integrity of the RNA and total RNA was quantified using Qubit 2.0 (Thermo Fisher Scientific Inc.).

Small RNA sequencing

Total RNA was subjected to multiplexed small RNA cDNA library preparation. Library preparation entails ligation of barcoded 3' adapters to 20 different samples, pooling of samples, ligation of a 5' adapter, reverse transcription and polymerase chain reaction (PCR), as previously described [18], with modifications allowing multiplexing of several 20-sample libraries on a single HiSeq lane, namely, 40-100 small RNA libraries per lane (see supplementary methods). Libraries were sequenced on an Illumina HiSeq sequencer, and the information obtained was analyzed by an automated computer pipeline to decode and annotate small RNA reads [19]. Normalization of miRNA reads was performed by dividing each miRNA read frequency by the total number of miRNA sequence reads within the subsample, thereby correcting the variable sequencing depth in each subsample. Real time PCR

Mature miRNAs were quantified using Perfecta® micro RNA Assays (Quanta Biosciences), according to the manufacturer's instructions. Real-time polymerase chain reaction (PCR) was performed on a StepOne real-time reverse transcription (RT)-PCR (Life Technologies) in triplicate for each sample. The fold changes of miRNAs were normalized to RNU6B, (ACT). The data is presented as 2^"ACT. Primer sequences were designed based on the miRNA sequences obtained from the miRBase database (http://microrna.sanger.ac.uk/). Statistical significance was calculated using 2-tailed t test.

Statistical analysis

Statistical procedures on count data were based on DESeq2, a publically available R/Bioconductor package for analysis of differential expression in RNA sequencing experiments [20], as previously described [21].

Example 1 - Identification of differentially expressed microRNAs before and after rituximab treatment and between Healthy Controls (HCs) and patients with NMO. A study was performed to identify miRNAs that are differentially expressed before and after rituximab treatment. Rituximab being a therapy for patients with NMO, with a response rate of approximately 90% [22, 23].

Blood samples were collected from Nine (9) NMO patients, classified as rituximab responders, prior to and at 6 months following therapy. Blood samples were also collected from Fifteen (15) age-and sex-matched Healthy subjects (HCs). To further analyze the differential expression between untreated NMOs and HCs, Seven (7) additional untreated AQP4+ seropositive NMO patients were also included in the study. Total RNA was extracted from the whole blood samples and miRNA levels were quantified by deep sequencing analysis (RNAseq).

RNAseq results demonstrated that the expression levels of 32 miRNAs were decreased and of 14 miRNAs were increased significantly (p<0.05) in the 9 NMO treated patients following treatment with rituximab. The results are presented in Table 1A. These nine (9) samples from responders were further compared with those from 5 non-responders. Using real time RT-PCR it was shown that miR-125 was significantly different for rituximab responders and non-responders (p=0.03). The additional 8 miRNAs that were analyzed were differently expressed in non- responders. Data from the 3 most significant miRNAs are presented in Fig. 1.

Analysis of the differential expression between the 16 untreated NMO patients and 15 HCs demonstrated that expression levels were significantly lower for 25 miRNAs and significantly higher for 17 miRNAs in NMO patients compared with HCs, as shown in Table IB.

Notably, the expression levels for 10 out of the 17 miRNAs with significantly increased expression in NMO patients reverted to the level of HCs following therapy with rituximab, as shown in Table 1A, and Fig. 2 A. In addition, miR-7 and miR-124, which had lower expression in NMO patients, reverted to the level of HCs following therapy with rituximab. The results are shown in Table 1A, and Fig. 2B.

The results presented herein show that 14 miRNAs were up regulated and 32 were down regulated significantly in the blood of NMO patients following effective therapy with rituximab (all p<0.05). Furthermore, it is shown that expression of 17 miRNAs was significantly higher and of 25 miRNAs was significantly lower in untreated NMO patients compared with healthy subject (HCs) (all p<0.05). Following rituximab treatment, the expression levels of 10 of the 17 miRNAs that show increased expression in NMO reverted to the levels seen in HCs. Six of these "normalized" miRNAs are known as brain specific/enriched miRNAs.

Tables 1A-1B: Differential expression of miRNAs following rituximab therapy (Table 1A) and between untreated NMO patients and HCs (Table IB). miRNA with increased expression in NMO patients that were normalized following rituximab therapy, indicated by (#); brain specific/enriched miRNAs are marked with (^Λ)

Table IB- Differential expression of miRNAs between untreated NMO patients and Healthy control subjects (HCs)

Example 2 - Identification of differentially expressed microRNAs before and after treatment and between Healthy Controls (HCs) and patients with MS.

A study is performed to identify miRNAs that are differentially expressed before and after suitable treatment (Rituximab, natalizuma and/or ocrelizumab). Blood samples were collected from MS patients, classified as treatment responders, prior to and at 6 months following therapy. Blood samples are also collected from age-and sex-matched Healthy subjects (HCs). To further analyze the differential expression between untreated MS patients and HCs, additional untreated seropositive MS patients are also included in the study. Total RNA is extracted from the whole blood samples and miRNA levels are quantified by deep sequencing analysis (RNAseq).

References

1. Wingerchuk, D.M., et al., The spectrum of neuromyelitis optica. Lancet Neurol, 2007. 6(9): p. 805-15.

2. Lennon, V.A., et al., IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med, 2005. 202(4): p. 473-7.

3. Sheinerman, K.S. and S.R. Umansky, Circulating cell-free microRNA as biomarkers for screening, diagnosis and monitoring of neurodegenerative diseases and other neurologic pathologies. Front Cell Neurosci, 2013. 7: p. 150.

4. Baumann, V. and J. Winkler, miRNA-based therapies: strategies and delivery p tforms for oligonucleotide and non-oligonucleotide agents. Future Med

Chem, 2014. 6(17): p. 1967-84.

5. Zernecke, A., et al., Delivery of microRNA- 126 by apoptotic bodies induces

CXCL12 -dependent vascular protection. Sci Signal, 2009. 2(100): p. ra81. 6. Valadi, H., et al., Exo some-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol, 2007. 9(6): p. 654-9.

7. Arroyo, J.D., et al., Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A, 2011. 108(12): p. 5003-8.

8. Vickers, K.C., et al., MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol, 2011. 13(4): p. 423- 33.

9. Keller, A., et al., Next-generation sequencing identifies altered whole blood microRNAs in neuromyelitis optica spectrum disorder which may permit discrimination from multiple sclerosis. J Neuroinflammation, 2015. 12: p. 196.

10. Trebst, C, et al., Update on the diagnosis and treatment of neuromyelitis optica: recommendations of the Neuromyelitis Optica Study Group (NEMOS). J Neurol, 2014. 261(1): p. 1-16.

11. Kimbrough, D.J., et al., Treatment of Neuromyelitis Optica: Review and

Recommendations. Mult Scler Relat Disord, 2012. 1(4): p. 180-187.

12. Papadopoulos, M.C., J.L. Bennett, and A.S. Verkman, Treatment of neuromyelitis optica: state-of-the-art and emerging therapies. Nat Rev

Neurol, 2014. 10(9): p. 493-506.

13. Huh, S.Y., et al., Mycophenolate mofetil in the treatment of neuromyelitis optica spectrum disorder. JAMA Neurol, 2014. 71(11): p. 1372-8.

14. Jacob, A., et al., Treatment of neuromyelitis optica with rituximab: retrospective analysis of 25 patients. Arch Neurol, 2008. 65(11): p. 1443-8.

15. Bedi, G.S., et al., Impact of rituximab on relapse rate and disability in neuromyelitis optica. Mult Scler, 2011. 17(10): p. 1225-30.

16. Jarius, S., et al., Antibody to aquaporin-4 in the long-term course of neuromyelitis optica. Brain, 2008. 131(Pt 11): p. 3072-80.

17. Wingerchuk, D.M., et al., Revised diagnostic criteria for neuromyelitis optica.

Neurology, 2006. 66(10): p. 1485-9.

18. Williams, Z., et al., Comprehensive profiling of circulating microRNA via small RNA sequencing of cDNA libraries reveals biomarker potential and limitations. Proc Natl Acad Sci U S A, 2013. 110(11): p. 4255-60. Farazi, T.A., et al., Bioinformatic analysis of barcoded cDNA libraries for small RNA profiling by next- generation sequencing. Methods, 2012. 58(2): p. 171-87.

Love, M.I., W. Huber, and S. Anders, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol, 2014. 15(12): p. 550.

Ben-Dov, I.Z., et al., Cell and Microvesicle Urine microRNA Deep

Sequencing Profiles from Healthy Individuals: Observations with Potential

Impact on Biomarker Studies. PLoS One, 2016. 11(1): p. e0147249.

Kim, S.H., et al., A 5 -year follow-up of rituximab treatment in patients with neuromyelitis optica spectrum disorder. JAMA Neurol, 2013. 70(9): p. 1110-

7.

Tobin, W.O. and S.J. Pittock, Rituximab Therapy in Neuromyelitis Optica: Moving Toward a Personalized Medicine Approach. JAMA Neurol, 2015. 72(9): p. 974-7.

Sempere, L.F., et al., Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biol, 2004. 5(3): p. R13.

Lagos-Quintana, M., et al., Identification of tissue-specific microRNAs from mouse. Curr Biol, 2002. 12(9): p. 735-9.

Miska, E.A., et al., Microarray analysis of microRNA expression in the developing mammalian brain. Genome Biol, 2004. 5(9): p. R68.

Landgraf, P., et al., A mammalian microRNA expression aths based on small RNA library sequencing. Cell, 2007. 129(7): p. 1401-14.

Leidinger, P., et al., The human miRNA repertoire of different blood compounds. BMC Genomics, 2014. 15: p. 474.

Cubillos-Ruiz, J.R., et al., Reprogramming immune responses via microRNA modulation. Microrna Diagn Ther, 2013. 1(1).

Shen, N., et al., MicroRNAs— novel regulators of systemic lupus erythematosus pathogenesis. Nat Rev Rheumatol, 2012. 8(12): p. 701 -9.

Gandhi, R., miRNA in multiple sclerosis: search for novel biomarkers. Mult Scler, 2015. 21(9): p. 1095-103.

Junker, A., et al., MicroRNA profiling of multiple sclerosis lesions identifies modulators of the regulatory protein CD47. Brain, 2009. 132(Pt 12): p. 3342- 52.

Ma, X., et al., Expression, regulation and function of microRNAs in multiple sclerosis. Int J Med Sci, 2014. 11(8): p. 810-8.

Keller, A., et al., Comprehensive analysis of microRNA profiles in multiple sclerosis including next- generation sequencing. Mult Scler, 2014. 20(3): p. 295-303.

Waschbisch, A., et al., G tiramer acetate treatment normalizes deregulated microRNA expression in relapsing remitting multiple sclerosis. PLoS One,

2011. 6(9): p. e24604.

Hecker, M., et al., MicroRNA expression changes during interferon-beta treatment in the peripheral blood of multiple sclerosis patients. Int J Mol Sci, 2013. 14(8): p. 16087-110.

Sievers, C, et al., Altered microRNA expression in B lymphocytes in multiple sclerosis: towards a better understanding of treatment effects. Clin Immunol,

2012. 144(1): p. 70-9. Ingwersen, J., et al., Natalizumab restores aberrant miRNA expression profile in multiple sclerosis and reveals a critical role for miR-20b. Ann Clin Transl Neurol, 2015. 2(1): p. 43-55.

Chen, C.Z., et al., Regulation of immune responses and tolerance: the microRNA perspective. Immunol Rev, 2013. 253(1): p. 112-28.

Head, S.R., et al., Library construction for next- generation sequencing: overviews and challenges. Biotechniques, 2014. 56(2): p. 61-4, 66, 68, passim. Duroux- Richard, I., et al., Circulating miRNA-125b is a potential biomarker predicting response to rituximab in rheumatoid arthritis. Mediators Inflamm, 2014. 2014: p. 342524.

Kim, S.H., et al., Repeated treatment with rituximab based on the assessment of peripheral circulating memory B cells in patients with relapsing neuromyelitis optica over 2 years. Arch Neurol, 2011. 68(11): p. 1412-20. Kim, S.H., et al., Treatment Outcomes With Rituximab in 100 Patients With Neuromyelitis Optica: Influence of FCGR3A Polymorphisms on the

Therapeutic Response to Rituximab. JAMA Neurol, 2015. 72(9): p. 989-95. Xinting M., et.al. Expression, Regulation and Function of MicroRNAs in Multiple Sclerosis. Int J Med Sci. 2014; 11(8): 810-818.

Claims

1. A method for determining efficacy of treatment for neuromyelitis optica (NMO) in a subject in need thereof, said method comprising:

a) determining the levels of at least two circulating miRNAs, selected from the group consisting of miR-138, miR-660, miR-125b, miR-760, miR- 100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180, in a first serum sample of said subject;

b) treating said subject with a selected treatment;

2. The method of claim 1, comprising determining the levels of at least three circulating miRNAs, selected from the group consisting of miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180.

3. The method of claim 1, wherein the selected treatment is selected from:

Rituximab, B cell depletion treatment, treatment with anti CD20, treatment with anti-CD19, ocrelizumab, ofatumumab (HuMax-CD20), Natalizumab, azathioprine, Cladribine, Lemtrada, and Terifiunomide.

4. The method of claims 1-3, wherein the selected treatment comprises Rituximab.

5. The method of claims 1-4, wherein, step a) is conducted at a time point prior to step b).

6. The method of claims 1-5, wherein steps a) and c) are conducted at distinct time points during step b).

7. The method of claims 1-6 further comprising repeating step c) for one or more times to determine the levels of the at least two circulating miRNAs in consecutive serum samples, obtained at designated time intervals; and comparing the levels of the at least two circulating miRNAs between said consecutive serum samples, wherein a modulation in the levels of the at least two circulating miRNAs between consecutive serum samples is indicative of said treatment efficacy.

8. The method of claim 1-7, wherein modulation comprises decrease in the level of the at least two miRNAs.

9. The method of claims 1-8, further comprising a step of isolating RNA from the first serum sample and/or the second serum prior to identification of the at least two miRNAs.

10. The method of claims 1-9, wherein the level of the at least two miRNAs in the first serum sample and/or the second serum sample is determined by a method selected from: amplification reaction, sequencing reaction, microarray, or combinations thereof.

11. The method of claims 1-10, wherein the treatment regime using the selected treatment is adjusted based on the determined treatment efficacy.

12. The method of claims 1-12 further comprising a step of comparing the levels of the at least two circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of corresponding miRNAs obtained from healthy control subjects.

13. A method for assessing treatment efficacy of a selected treatment in a subject afflicted with neuromyelitis optica (NMO), the method comprising determining the level of at least two circulating miRNA molecules, selected from the group consisting of: miR-138, miR-660, miR-125b, miR-760, miR- 100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR- 1180, in serum samples of said subjects obtained before and after treatment with the selected treatment, and comparing the levels of the at least two circulating miRNA molecules between the serum samples, wherein modulation of expression of the miRNA molecules between the serum samples obtained before and after treatment is indicative of increased treatment efficacy.

14. The method of claim 12, wherein the treatment is selected from: Rituximab, B cell depletion treatment, treatment with anti CD20, treatment with anti-CD19, ocrelizumab, ofatumumab (HuMax-CD20), Natalizumab, azathioprine, Cladribine, Lemtrada, Teriflunomide, and combinations thereof.

15. The method of claims 13-14, wherein the selected treatment comprises Rituximab.

16. The method of claims 13-15, further comprising comparing the at least two circulating miRNA molecules between the consecutive serum samples.

17. The method of claims 13-16, wherein RNA is isolated from the serum samples prior to identification of the at least two miRNAs.

18. The method of claims 13-17, wherein the level of miRNA in the serum is determined by a method selected from: amplification reaction, sequencing reaction, microarray, or combinations thereof.

19. The method of claims 13-18, further comprising evaluating the treatment efficacy by clinical parameters, including determining the level of anti Aquaporin 4 expression levels.

20. The method of claims 13-19, wherein said modulation comprises increased levels of the miRNAs and/or reduced levels of the miRNAs.

21. The method of claims 13-20, further comprising a step of comparing the levels of circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of miRNAs obtained from healthy control subjects.

22. A kit for determining efficacy of a selected treatment for neuromyelitis optica

(NMO), comprising means for determining the levels of at least two circulating miRNAs, selected from: miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180 in serum samples of a subject, said samples obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for the neuromyelitis optica (NMO).

23. The kit according to claim 22, wherein the selected treatment comprises:

Rituximab, B-cell depletion treatment, treatment with anti CD20, treatment with anti-CD19, ocrelizumab, ofatumumab (HuMax-CD20), Natalizumab, Azathioprine, Cladribine, Lemtrada, Teriflunomide, and combinations thereof.

24. The kit according to claims 22-23, wherein the selected treatment comprises:

Rituximab

25. The kit according to claim 22, wherein the means comprises specific nucleic acid molecules for identification of circulating miRNAs in the serum samples.

26. The kit according to claim 25, wherein the nucleic acid molecules comprise specific primers for identification of miRNAs in an amplification reaction performed on RNA isolated from the serum sample.

27. The kit according to claims 25-26, wherein the nucleic acid molecules comprise specific probes for identification of circulating miRNAs in a sequencing reaction performed on RNA or DNA isolated from the serum sample.

28. The kit according to claims 22-27, wherein the instructions further comprising comparing the levels of circulating miRNAs in the serum samples of said subject to the circulating levels of miRNAs obtained from healthy control subjects.

29. A method for determining treatment efficacy of multiple sclerosis (MS) in a subject in need thereof, said method comprising:

b) treating said subject with a selected treatment;

30. The method of claim 1, wherein the selected treatment is selected from:

Rituximab, B cell depletion treatment, treatment with anti CD20, treatment with anti-CD19, ocrelizumab, ofatumumab (HuMax-CD20), Natalizumab, azathioprine, Cladribine, Lemtrada, and Teriflunomide.

31. The method of claims 29-30, wherein, step a) is conducted at a time point prior to step b).

32. The method of claims 29-31, wherein steps a) and c) are conducted at distinct time points during step b).

33. The method of claims 29-31 further comprising repeating step c) for one or more times to determine the levels of the at least two circulating miRNAs in consecutive serum samples, obtained at designated time intervals; and comparing the levels of the at least two circulating miRNAs between said consecutive serum samples, wherein a modulation in the levels of the at least two circulating miRNAs between consecutive serum samples is indicative of said treatment efficacy.

34. The method of claim 29-33, wherein modulation comprises decrease or increase in the level of the at least two miRNAs.

35. The method of claims 29-34, wherein the treatment regime using the selected treatment is adjusted based on the determined treatment efficacy.

36. The method of claims 29-35, further comprising a step of comparing the levels of the at least two circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of corresponding miRNAs obtained from healthy control subjects.

37. A method for assessing treatment efficacy of a selected treatment in a subject afflicted with Multiple Sclerosis (MS), the method comprising determining the level of at least two circulating miRNA molecules, selected from the group consisting of: miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180, in serum samples of said subjects obtained before and after treatment with the selected treatment, and comparing the levels of the at least two circulating miRNA molecules between the serum samples, wherein modulation of expression of the miRNA molecules between the serum samples obtained before and after treatment is indicative of increased treatment efficacy.

38. The method of claim 37, wherein the treatment is selected from: Rituximab, B cell depletion treatment, treatment with anti CD20, treatment with anti-CD19, ocrelizumab, ofatumumab (HuMax-CD20), Natalizumab, azathioprine,

Cladribine, Lemtrada, Teriflunomide, and combinations thereof.

39. The method of claims 37-38, further comprising comparing the at least two circulating miRNA molecules between the consecutive serum samples.

40. The method of claims 37-39, further comprising evaluating the treatment efficacy by clinical parameters, including determining the level of anti Aquaporin 4 expression levels.

41. The method of claims 37-40, wherein said modulation comprises increased levels of the miRNAs or reduced levels of the miRNAs.

42. The method of claims 37-41, further comprising a step of comparing the levels of circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of miRNAs obtained from healthy control subjects.

43. A kit for determining efficacy of a selected treatment for multiple sclerosis (MS), the kit comprises means for determining the levels of at least two circulating miRNAs, selected from: miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180 in serum samples of a subject, said samples obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for the multiple sclerosis (MS).

44. The kit according to claim 43, wherein the selected treatment comprises:

45. The kit according to claims 43-44, wherein the selected treatment comprises:

Rituximab

46. The kit according to claim 43, wherein the means comprises specific nucleic acid molecules for identification of circulating miRNAs in the serum samples.

47. The kit according to claim 46, wherein the nucleic acid molecules comprise specific primers for identification of miRNAs in an amplification reaction performed on RNA isolated from the serum sample.

48. The kit according to claims 46-47, wherein the nucleic acid molecules comprise specific probes for identification of circulating miRNAs in a sequencing reaction performed on RNA or DNA isolated from the serum sample.

49. The kit according to claims 43-48, wherein the instructions further comprising comparing the levels of circulating miRNAs in the serum samples of said subject to the circulating levels of miRNAs obtained from healthy control subjects.