WO2019116412A1 - Method for in vitro diagnosis of amyotrophic lateral sclerosis - Google Patents

Abstract

The present invention concerns a method for in vitro diagnosis of 5 Amyotrophic Lateral Sclerosis by the detection of exosomal RNAs in a biological sample such as cerebrospinal fluid (CSF).

Description

Method for in vitro diagnosis of Amyotrophic Lateral Sclerosis

The present invention concerns a method for in vitro diagnosis of Amyotrophic Lateral Sclerosis (ALS). Particularly, the present invention concerns a method for in vitro diagnosis of Amyotrophic Lateral Sclerosis by the detection of exosomal mRNAs in a biological sample such as cerebrospinal fluid (CSF).

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the degeneration of upper and lower motor neurons, leading to muscle weakness, atrophy and progressing to complete paralysis. ALS inevitably evolves to death and its impact on the quality of life is devastating. Two to four people per 100,000 develop the disease each year (Turner and Talbot, 2008). ALS may occur in two forms, sporadic (sALS) and familial (fALS). A genetic etiology has been identified in both types, specifically in up to 1 1 % of apparently sALS and 68% of fALS cases, with at least 16 genes and genetic loci being implicated in ALS pathogenesis (Renton et al., 2014; Vucic et al., 2014).

Nearly 12% of FALS cases are linked to a mutation in the Cu/Zn superoxide dismutase (SOD7), responsible for scavenging free radicals. More than one hundred mutations of the SOD1 gene have been identified so far, all showing dominant hereditary patterns. Although many molecular mechanisms have been proposed to drive ALS pathogenesis, 18 years after the initial discovery no consensus has yet emerged as to the toxicity of mutant SOD1{ Rosen et al., 1993). Mutations in other genes, that cause familial ALS or contribute to the development of sporadic ALS, have been identified. TARDBP gene mutations account for about 4% of fALS cases; FUS gene mutations cause about 4% (Renton et al., 2014). It is estimated that 68% of individuals with familial ALS have an identified genetic mutation. The cause of the condition in the remaining individuals remains unknown (Renton et al., 2014)(Vucic et al., 2014).

A major advance in the understanding of ALS pathogenesis occurred with the discovery of the dominantly inherited C9orf72 gene, which appears to underlie over 40% of fALS and 7% of sALS cases (Renton et al., 2014)(DeJesus-Hernandez et al., 201 1 ). This finding is a milestone in ALS research and has greatly contributed to the understanding of ALS (Majounie et al., 2012).

Nevertheless both therapy and diagnosis for this motor neuron disease are still challenging. Molecular diagnostic tests are surprisingly still missing with the exception of mutations of identified genes in a limited number of fALS patients. Neurologists and specialists in neuromuscular diseases both claim difficulties with early diagnosis of ALS. The revised El Escorial Criteria edited in 2000, state that to establish the diagnosis of ALS a combination of lower and upper motoneuron signs with evidence of spread is required. There was, however, a consensus among researchers that early biomarkers are urgently needed to facilitate ALS diagnosis and prognosis and improving patient care as well as to act as indicators of therapeutic response in clinical trials (Silani et al., 201 1 ).

In the light of the above, it is therefore apparent the need to provide for methods of in vitro diagnosis of ALS.

In recent years, many publications have shown that exosomal vesicles released by most cell types can be used as precise diagnostic tools in a number of pathologies including certain tumours and some neurodegenerative diseases including Alzheimer diseases (AD) (Properzi et al., 2013).

Exosomes represent a specific subtype of secreted nanovesicles (50-90 nm in diameter) that are formed in internal endosomal compartments and with various functions (EL Andaloussi et al., 2013) (Logozzi et al., 2009; Properzi et al., 2013). They are important intercellular communicators shuttling molecular and genetic information from one cell to another. Their molecular content is highly selected and includes specific lipids and proteins. In addition, in 2007, a milestone publication by Valadi and colleagues revealed the presence of RNA species in human and mouse mast cell exosomes that are delivered and functional in target cells (Valadi et al., 2007). These RNAs have been called exosomal shuttle RNAs (esRNA) and include mRNA encoding up to 1300 genes and various micro RNA (miRNA) species. As exosomes are released in easily accessible body fluids, their content represents a precious biomedical tool which is used for the diagnosis and prognosis of an increasing number of pathologies including neurodegenerative diseases (Properzi et al., 2013)(Coleman and Hill, 2015). Interestingly the majority of the misfolded proteins involved in neurodegeneration, including SOD1 (Gomes et al., 2007), are released in association with exosomes and the detection of microRNA associated with AD in biological fluids using next-generation sequencing technologies was recently also shown (Cheng et al., 2013). Exosomes miRNA profiling of the serum of AD patients at early stages of the pathology revealed a specific signature of 16 AD-specific deregulated miRNAs (Cheng et al., 2014).

Several studies have examined miRNAs in ALS models and in patient biopsy samples. In a recent work, done by Freischmidt and colleagues (Freischmidt et al., 2013), it is reported that in patients with sALS, the expression of certain miRNAs is altered in CSF and in serum (miR-132-5p, miR-132-3p and miR-143-3p, miR -143-5p and miR-574- 5p). miRNAs were previously shown to bind to TDP-43 in vitro (Freischmidt et al., 2013).

Using G93A-SOD1 transgenic mice as the mouse model of ALS, it was seen that the expression of miR-206, which plays a role in skeletal muscle development, increases significantly in synaptic regions of muscle fibres of the mouse model of ALS (Williams et al., 2009). The levels of this miRNA are also increased systemically in both mice and humans (Toivonen et al., 2014).

Important results that strengthen the role of miRNAs in the pathogenesis of ALS and confirm them as possible candidates for diagnostic markers, are described by Parisi and colleagues (Parisi et al., 2013) who performed a comparative screening of miRNAs in resting and activated SOD1 -G93A microglia and identified selected miRNAs (miR-22, miR-155, miR-125b and miR-146b) to be used as novel tools for further dissecting and controlling mechanisms. These results support the idea that ALS is a neuroinflammatory disease, as they identified specific miRNAs that operate by modulating ALS-linked inflammatory genes and suggested their deregulation as pathogenetic mechanisms of the disease (Parisi et al., 2013).

miR-124 was previously shown to be secreted by neuronal cell cultures and internalized by astrocytes. Interestingly, exogenous delivery of miR-124a in vivo through stereotaxic injection significantly prevents further pathological loss of GLT1 proteins, as determined by GLT1 immunoreactivity in SOD1 G93A mice (Morel et al., 2013).

According to the present invention it has been now found that specific exosomal RNAs can be advantageously used as biomarkers for the in vitro diagnosis of ALS.

According to the present invention a method for the isolation of the exosomes from CSF samples was firstly optimized. Subsequently a microarray screening analysis of miRNA and mRNA was performed on 15 sporadic ALS patient samples and 15 relevant neurological controls. Data analysis revealed that 16 miRNAs and 7 mRNAs were significantly deregulated in patients affected by ALS, compared to controls. These data were further validated by RT-PCR on the same exosome samples confirming the results of the initial screening. Importantly one of the miRNAs, miR-34b-3p, and two of the mRNAs, SOD2, SLC25A21 , were found exclusively present in sporadic ALS patients and therefore specific to the pathology. When diagnostic accuracy was calculated considering the three hits together, a specificity of 100% and sensitivity of 93% was estimated. These impressive data are supported by the fact that the three biomarkers are all part of the same cellular pathway and they are possibly involved in the pathogenesis of ALS.

The finding of novel ALS-associated RNAs in body fluid exosomes allows an innovative and low cost approach for diagnostic purposes and provide insights on the pathogenesis of this fatal diseases that is still lacking accurate diagnosis and cure.

None of the RNAs according to the present invention were previously shown to be associated with ALS.

It is interesting that one of the deregulated mRNA, which is selectively expressed in ALS samples is SOD2, a member of the iron/manganese superoxide dismutase family. The protein transforms toxic superoxide, product of the mitochondrial electron transport chain, into hydrogen peroxide and diatomic oxygen. This function allows SOD2 to clear mitochondrial reactive oxygen species (ROS) and, as a result, confer protection against cell death. As a result, it plays an anti-apoptotic role against oxidative stress, ionizing radiation, and inflammatory cytokines. Mutations in this gene have been associated with idiopathic cardiomyopathy (IDC), premature aging, sporadic motor neuron disease, and cancer (Zou et al., 2017). In addition to SOD2, the mRNA SLC25A21 , a mitochondrial 2-oxodicarboxylate carrier, was also selectively secreted on CSF exosomes of some patients and miR-34b-3p. Interestingly, this small RNA is upregulated in miotonic dystrophy type-2 in biopsies of skeletal muscle (Greco et al., 2012). Deregulation of miR-34b and c, which are direct targets of p53, in PD triggers downstream transcriptome alterations underlying mitochondrial dysfunction and oxidative stress, which ultimately compromise cell viability (Mihones-Moyano et al., 201 1 ).

Mitochodrial pathway is therefore related with the most significant hits identified by the present work, indicating the involvement of this cellular compartment in ALS diseases, possibly in its pathogenesis as it was previously described (Smith et al., 2017).

It is therefore a specific object of the present invention a method for in vitro diagnosis of Amyotrophic Lateral Sclerosis, said method comprising the detection, in a biological sample, of all, two or at least one of the following exosomal RNAs: miR-34b-3p, SOD2, SLC25A21 , wherein said RNAs are expressed (i.e. they are present and therefore detected) in Amyotrophic Lateral Sclerosis.

According to an embodiment of the present invention, the method can comprise:

a)extracting exosomes in a biological sample ;

b)extracting RNAs from exosomes;

c)detecting all, two or at least one of the following exosomial RNAs: miR-34b-3p, SOD2, SLC25A21 , wherein said RNAs are present in Amyotrophic Lateral Sclerosis.

According to a further embodiment of the present invention, the method can further comprise the detection, in a biological sample, of all, more than one or one of the following exosomal mirRNAs: miR-335-5p, miR-99b-5p, miR-136-5p, miR-130b-3p, miR-326, wherein said miR-335- 5p, miR-99b-5p, miR-136-5p, miR-130b-3p, miR-326 are expressed in healthy subjects.

According to a further embodiment of the present invention, the method can further comprise the detection, in a biological sample, of all, more than one or one of the following exosomal mirRNAs: miR-124-3p, miR-222-3p, miR-143-3p, let-7b-5p, let-7d-3p, miR-145-5p, let-7i-5p, miR- 9-3p, miR-15a-5p, let-7a-5p, wherein miR-124-3p, miR-222-3p, miR-143- 3p, let-7b-5p, let-7d-3p, miR-145-5p, let-7i-5p, miR-9-3p, miR-15a-5p are downregulated in Amyotrophic Lateral Sclerosis in comparison with healthy controls, whereas let-7a-5p is upregulated in Amyotrophic Lateral Sclerosis in comparison with healthy controls.

According to a further embodiment of the present invention, the method can further comprise the detection, in a biological sample, of all, more than one or one of the following exosomal mRNAs: SLC25A3, CPU B, OPA1 , SLC25A16 and SLC25A24, wherein SLC25A3 and CPU B are upregulated in Amyotrophic Lateral Sclerosis in comparison with healthy controls, whereas OPA1 , SLC25A16 and SLC25A24 are downregulated in Amyotrophic Lateral Sclerosis in comparison with healthy controls.

According to the present invention, the biological sample is a liquid biological sample such as blood, nasal lavage, supernatant of cells extracted from biological tissues.

The method according to the present invention can be carried out for example by Real Time PCR, Droplet Digital PCR, Microarray, RNA Hybridization Methods such as Northern Blot or Dot Blot, RNA Next Generation Sequencing.

The present invention concerns also the use of all, more than one or at least one of the following RNAs as biomarkers for the in vitro diagnosis of Amyotrophic Lateral Sclerosis: miR-34b-3p, SOD2, SLC25A21 , miR-335-5p, miR-99b-5p, miR-136-5p, miR-130b-3p, miR- 326, miR-124-3p, miR-222-3p, miR-143-3p, let-7b-5p, let-7d-3p, miR-145- 5p, let-7i-5p, miR-9-3p, miR-15a-5p, let-7a-5p, SLC25A3, CPT1 B, OPA1 , SLC25A16 or SLC25A24.

The present invention now will be described by an illustrative, but not limitative way, according to preferred embodiments thereof, with particular reference to the enclosed drawings, wherein:

Figure 1 shows the miRNA screening: The CSF exosome miRNA content of 15 sporadic ALS samples and 15 neurological controls was analyzed by microarray. A) The cDNA synthesis control (UniSp6) was added in the reverse transcription reaction giving the opportunity to evaluate the RT reaction. In addition to this a DNA spike-in (UniSp3) is present on all panels. Both spike-ins displayed Cq well below 37. B) The box plot chart shows the 16 miRNAs that had significantly different values in sporadic ALS patients when compared to neurological controls. The lines in the middle of boxes represent the median value. The boxes extends from the 25th (x[25]) to the 75th (x[75]) percentile, bars (lines/wiskers) extends from x[25]-1.5 IQR to x[75] + 1.5 IQR where IQR is Interquartile range.

Figure 2 shows the mRNA screening: The CSF exosome mRNA content of 15 sporadic ALS samples and 15 neurological controls was analyzed using a mitochondrial array. A) The box plot chart shows that 5 mRNAs had significantly different values in sporadic ALS patients when compared to neurological controls. The lines in the middle of boxes represent the median value. The boxes extends from the 25th (x[25]) to the 75th (x[75]) percentile, bars (lines/wiskers) extends from x[25]-1.5 IQR to x[75] + 1.5 IQR where IQR is Interquartile range. B) Two mRNA (SOD2 and SLC25A21 ) were selectively present in the ALS exosomes in 47% and 53% of patients.

Figure 3 shows the Diagnostic accuracy of ALS-specific esRNAs: The sensitivity and specificity was calculated for three biomarkers that were selectively expressed in ALS patients. A) As shown in the bar chart miR-34-3p and S0D2 had a sensitivity of 27% and 53% respectively and a specificity of a 100%, while SLC25A21 sensitivity was 47% and specificity 93%. The specificity increased to 100% when it was calculated considering the three RNAs. B) No correlation was found in between the type of biomarker and the age, sex or onset of diseases.

EXAMPLE 1 : Study of a novel exosomal shuttle RNA for the diagnosis of Amyotrophic Lateral Sclerosis

MATERIALS AND METHODS

CSF sample collection and storage

All patients who participated in the study belong to Ospedale San Luca - IRCCS Istituto Auxologico Italiano (Milan). The cohort included 15 (screening) + 66 (validation) ALS patient of Italian descent (Tablel ). All of them received a diagnosis of probable or definite sporadic ALS, according to the El Escorial revised criteria at a tertiary care ALS Center. A panel of 15 (screening) + 28 (validation) control individuals with neurological diseases (Table 1) was used for comparison of CSF parameters.

We received approval from the ethical standards committee on human experimentation of the IRCCS Istituto Auxologico Italiano. Written informed consent was obtained from all patients participating in the study (consent for research).

Lumbar puncture was performed in all patients as part of the routine diagnostic procedures after they had given a written informed consent. Localized intervertebral space L3-L4 and L4-L5, it numbs the skin with a local anesthetic spray (dry ice, ethyl chloride), with the patient recumbent on his side in a fetal position. It uses a needle by lumbar puncture with the spindle, a traumatic (with tip Quincke), generally 19G in diameter. Once positioned the needle and remove the chuck, it let it drip CSF in polypropylene tubes (4-6 cc of total liquor, for diagnostic and conservation). Approximately 8 hours after the picking, samples were stored at + 4 ° C until analysis (for diagnostics) or storage in the biobank at -80 ° C, all in polypropylene tubes.

Table 1 ALS patient Control patient diagnosis diagnosis

Discovery 15 sporadic 5 Alzheimer disease

phase 5 Creutzfeldt-Jakob

disease

1 Neurodegenerative dementia

1 Lewy body dementia

1 Monoparesis

2 Polyneuropathy

Validation 66 sporadic Alzheimer disease, phase Multiple sclerosis,

neurodegenerative

dementia, cognitive impairment

Exosome isolation

CSF (0.250 ml) samples were thawed at room temperature and centrifuged at 2,300 c g for 30 min to remove cell debris. Exosomes isolation was performed as described by Logozzi et al. (Logozzi et al., 2009). Briefly supernatants were filtered using a 0.22 pm filter (Millipore Corp., Bedford, MA) and centrifuged at 100,000 g for 1 hour in a Beckman ultracentrifuge (Beckman Coulter) in order to pellet exosomes. After 1 wash in a large volume of phosphate-buffered saline (PBS), exosomes were resuspended in PBS or lysis buffer (20 pi) and stored at -80 °C for experimental analysis.

Protein quantification and Western Blots

Exosomes purified by ultracentrifugation were treated with lysis buffer (1 % Triton X-100, 0,1 % SDS, 0.1 M Tris HCI, pH 7) and protease inhibitors (Sigma) and protein concentration was determined by Bradford microassay (Bio-Rad Laboratories, Hercules, CA). Proteins were separated on 10% SDS-PAGE gel and transferred to nitrocellulose membranes. Membranes were blotted with antibodies to CD63 (Mabs, Pharmingen), Rab-5b (polyclonal antibody, Santa Cruz), incubated with appropriate HRP-conjugated secondary antibodies (Amersham Pharmacia) and visualised by enhanced chemiluminescence (Pierce).

RNA extraction

Total RNA was extracted with miRNeasy Mini Qiagen kit. Briefly, the exosomes (10 mI) were lysed in 700 mI of Qiazol lysis buffer and homogenized by passing 10 times in the syringe. The resulting lysate was transferred to a new tube containing 140 mI of chloroform, mixed, incubated for 2 min at room temperature, and centrifuged at 12000 x g for 15 min at 4°C. The aqueous phase was transferred to a new tube containing 1.5 volumes of ethanol 100%. The content was mixed and transferred to Qiagen RNeasy Mini spin columns, washed and centrifuged following the manufacturer's protocol. Total RNA was finally eluted with 50 mI of RNase-free water and stored in five aliquots at -80 ° C until use. The amount of total RNA extracted was assessed with readings to Nanodrop 2000 (average 10.5 ng / mI).

Microarray miRNA screening (Discovery phase)

10 pi RNA was reverse transcribed in 50 mI reactions using the miRCURY LNA™ Universal RT microRNA PCR, Polyadenylation and cDNA synthesis kit (Exiqon). cDNA was diluted 50 x and assayed in 10 mI PCR reactions according to the protocol for miRCURY LNA™ Universal RT microRNA PCR; each microRNA was assayed once by qPCR on the microRNA Ready-to-Use PCR, Human panel I using ExiLENT SYBR® Green master mix. Negative controls excluding template from the reverse transcription reaction was performed and profiled like the samples. The amplification was performed in a LightCycler® 480 Real-Time PCR System(Roche) in 384 well plates. The amplification curves were analyzed using the Roche LC software, both for determination of Cq (by the 2nd derivative method) and for melting curve analysis. An additional step in the real-time PCR analysis was performed to evaluate the specificity of the amplification products by generating a melting curve for each reaction. The appearance of a single peak with the expected Tm is an indication that a single specific product was amplified during the qPCR process. PCR reactions that gave rise to multiple melting curve peaks or single peaks with melting temperature that was inconsistent with the specifications or the corresponding assay (in-house database) were flagged and removed from the dataset. A“no template” sample in the RT step was included as a negative control. An assay detected 5 Cqs lower than the negative control will be included in the data analysis. For assays that do not yield any signal on the negative control, the upper limit of detection is set to Cq=37. The cDNA synthesis control (UniSp6) was added in the reverse transcription reaction giving the opportunity to evaluate the RT reaction. In addition to this a DNA spike-in (UniSp3) is present on all panels. The DNA spike-in consists of a premixed combination of DNA template and primers. Deviations in this reaction indicate inhibitions at the qPCR level.

miRNA Real-Time PCR (Validation phase)

A total of 4 mI RNA (~ 40 ng) was used in 20 mI of reverse transcription according to the protocol of the kit miRCURY LNA Universal RT microRNA PCR Kit (Exiqon). The cDNA obtained was diluted 50 times and tested in 10 mI PCR with the ExiLENT SYBR Green master mix protocol and LNA microRNA PCR primers (miRNA of interest). The amplification was carried out in thermocycler Applied Biosystems 7500 in 96-well plates following the protocol supplied by Exiqon.

The RNA spike-in kit was used for RNA isolation and PCR technical control. A negative control was also included for each essay.

mRNA Screening and Real-Time PCR (Discovery and Validation)

A total of 4 mI RNA was used in 20 mI of reverse transcription according to the RT² First Strand Kit (Qiagen) protocol. Genomic DNA elimination step was performed according to the manufacturer's protocol. The resulting cDNA samples were added with 91 mI RNase-free water and processed by the real-time PCR protocol using the RT² Profiler mithocondrial PCR array (cat. No. PAFIS-087Z) with the RT² SYBR Green Mastermix (Qiagen). The mithocondrial array was chosen as it was considered the most relevant to the pathology. Negative, genomic contamination, and positive controls were included in the array. Validation of the obtained results was similarly performed with custom RT² qPCR primer assays (Qiagen).

Data analysis

The amplification curves were analyzed using the ABI7500 instrument software both to determining the cq and for the analysis of melting curves.

All assays presenting multiple melting temperatures or temperatures different from expected were omitted from the analysis.

Given the difficulty of using appropriate normalization in these experiments (specific for exosomes in CSF), for each samples the same volume of CSF for the purification of exosomes, the same volume of exosomes for the extraction of RNA, and the same volume of the resulting RNA for RT-PCR quantification were used. Moreover a qualitative analysis was also performed (absence or presence of the miRNA and mRNA). The miRNA spike-in was used to adjust differences in RNA extraction efficiency.

Comparisons among groups were carried out by Mann-Whitney U test for continuous variables and by Fisher’s exact probability test for categorized parameters (positive vs negative). Diagnostic accuracy was assessed by sensitivity, specificity and ROC curve analysis. The area under the ROC Curve (AUC) was used to measure the accuracy of the parameter in discriminating ALS vs Ctrl. Multivariate logistic regression was carried out to assess the independent role of multiple parameters.

RESULTS

CSF exosome isolation

To ascertain that exosomes could be successfully isolated from CSF samples a pool of control samples (2ml final volume) have been processed using a standard exosome isolation protocol. 50pg of proteins of resupended pellet were analyzed by Western Blots using anti-exosomal antibodies, as previously described. Immuno-positive bands were observed after incubation with antibodies against exosomal markers but not after incubation with antibodies against cellular markers confirming the presence and the successful isolation of nanovesicles in the pellets (data not shown).

miRNA Screening Results

The CSF of 15 sporadic ALS samples and 15 neurological controls was processed for exosome isolation. The pellet was re-suspended and the RNA extracted accordingly to the method described. The miRNA content was analyzed by microarray using the Human Panel I from Exiqon. The panel profiling was successfully completed and all the raw data showed good quality. No signal was detected on negative control samples and a solid positive signal was associated to both spike-ins (Figure 1 a). Of 383 total miRNA analyzed, 16 showed significantly different values in sporadic ALS patients (Figure 1 b).

Table 2 shows the list of the deregulated miRNA and the related p- value, which was obtained by using a Mann Whitney U test.

It is important to note that one of the miRNAs was detected only in ALS samples (miR-34b-3p) while five of them were detected only in controls (miR-335-5p, miR-99b-5p, miR-136-5p, miR-130b-3p, miR-326). The remaining miRNAs were expressed in both controls and ALS patients but the levels were found to be significantly different. Specifically miR-124- 3p, miR-222-3p, miR-143-3p, let-7b-5p, let-7d-3p, miR-145-5p, let-7i-5p, miR-9-3p, miR-15a-5p levels were lower in exosomes of ALS patients while, let-7a-5p were higher. miR-124-3p and mir-335-5p were the miRNA with the lowest p-value and therefore displaying a larger disparity between the two groups. Results were subsequently validated in the same samples by RT-PCR.

TABLE 2

Deregulated miRNAs in CSF exosomes of sporadic ALS patients miRNA miRBase p-value Exosome Regulation accession (Mann Whitney)

number

miR-124-3p MIMAT0000422 0.0024 Downregulated

0.0031 Expressed Only in miR-335-5p MIMAT0000765 Controls

miR-222-3p MIMAT0000279 0.0092 Downregulated miR-143-3p MIMAT0000435 0.0124 Downregulated let-7b-5p MIMAT0000063 0.0128 Downregulated let-7a-5p MIMAT0000062 0.0147 Upregulated

0.0160 Expressed Only in miR-99b-5p MIMAT0000689 Controls

0.0160 Expressed Only in miR-136-5p MIMAT0000448 Controls

let-7d-3p MIMAT0004484 0.0165 Downregulated miR-145-5p MIMAT0000437 0.0259 Downregulated miR-130b- 0.0346 Expressed Only in

3p MIMAT0000691 Controls

0.0346 Expressed Only in miR-326 MIMAT0000756 Controls

miR-34b-3p MIMAT0004676 0.0346 Expressed Only in ALS let-7i-5p MIMAT0000415 0.0352 Downregulated miR-9-3p MIMAT0000442 0.0416 Downregulated miR-15a-5p MIMAT0000068 0.0456 Downregulated

Analysis of dichotomized parameter (expressed versus non expressed) by Fisher’s exact probability test: p=0.049 mRNA Screening Results

The CSF of 15 sporadic ALS samples and 15 neurological controls was processed for exosome isolation. The pellet was re-suspended and the RNA extracted accordingly to the method described. The mRNA content was analyzed using a mitochondrial PCR array. The panel profiling was successfully completed and all the raw data showed good quality. No signal was detected on negative control samples. Of 96 total mRNA analyzed, 7 showed significantly different values in sporadic ALS patients (Figure 2). Table 3 shows the list of the deregulated mRNA and the related p-value, which was obtained by using a Mann Whitney U test. Two of the mRNAs were detected only in ALS samples (SOD2 and SLC25A21 ) (Figure 2a). The remaining five miRNAs were expressed in both controls and ALS patients but the levels were found to be significantly different (Figure 2b). Specifically SLC25A3 and CPT1 B were found to be upregulated in ALS samples, while OPA1 , SLC25A16, SLC25A21 and SLC25A24 where downregulated. As described in the method section, average values were considered significantly different in all the cases where the p-value was lower than 0.05. Nevertheless three of the identified mRNA, CPT1 B, SLC25A16 and OPA1 had a p-value respectively of 0.0649, 0.0712 and 0.0779 (Table 3). We decided to include them in the hit list due to low number of samples.

TABLE 3

Deregulated mRNAs in CSF exosomes of sporadic ALS patients mRNA GeneBank p-value ALS Exosome

(Mann Expression

Whitney)

SOD2 NM_00063 0.0034 Expressed Only in

6 ALS

SLC25 NM_01338 0.0171 Downregulated

A24 6

SLC25 NM_00263 0.0171 Upregulated

A3 5

SLC25 NM_03063 0.0265 Expressed Only in

A21 1 ALS

CPT1 B NM_00437 0.0649 Upregulated

7

SLC25 NM 15270 0.0712 Downregulated A16 7

OPA1 NM_13083 0.0779 Downregulated

_ 7 _

^* Analysis of dichotomized parameter (expressed versus non expressed) by Fisher’s exact probability test: p=0.002

^** Analysis of dichotomized parameter (expressed versus non expressed) by Fisher’s exact probability test:: p=0.035

Specificity and sensitivity of ALS associated RNAs

Biomarkers miR-34b-3p, SOD2, SLC25A21 were found to be selectively expressed in sporadic ALS exosomes. They were expressed respectively in 4, 8 and 7 patients out of 15 total. In the case of SLC25A21 , one control out of 15 total samples was found to be positive.

The diagnostic accuracy was calculated for each of the three biomarkers: miR-34-3p and SOD2 had a sensitivity of 27% and 53% respectively and a specificity of a 100%, while SLC25A21 sensitivity was 47% and specificity 93%.

Interestingly when the diagnostic accuracy was calculated considering the number of positive samples to any of the three biomarkers, the specificity increased to 100% (Figure 3a). No correlation was found in between the type of biomarker and the age, sex or onset of diseases (Figure 3b).

Validation of ALS-associated RNAs on a larger cohort of patients

The biomarkers that were selectively expressed in the CSF exosomes of ALS patients were further analyzed and validated on a larger cohort of patient. Exosomes were isolated from 66 CSF samples of ALS sporadic patients and 28 neurological controls. For each mRNA parameter (SOD2, SLC25A21 , and SLC25A3), the ROC curve was constructed, calculating the sensitivity and specificity at each outpoint. The area under the ROC Curve (AUC) was used to measure the accuracy of the parameter in discriminating ALS vs Ctrl. Fisher's exact test was applied to evaluate the difference between ALS and Ctrl in the proportion of subjects with parameter determined (<42) and undetermined (= 42). The independent role of each dichotomized parameter (> 42 vs 42) significant to univariate analysis in discrimination between ALS and Ctrl was evaluated by a multivariate logistic regression model. Accuracy of SOD2 determined (<42) was: sensitivity=23%, specificity=96%

Accuracy of SLC25A21 undetermined (=42) was: sensitivity=89%, specificity=42%.

Results of multivariate logistic regression analysis suggested that both SOD2 and SLC25A21 are significantly and independently associated to ALS. Indeed the only control subject with SOD2 determined presented SLC25A21 undetermined, so the combined criterion“SOD2 determined and SLC25A21 undetermined” reached specificity=100%. According to results of multivariate logistic regression analysis a probability of being SLA might be calculated according to results of SOD2 and SLC25A21 (Table 4).

Table 4

Validation of miRNA (Analysis carried out on subjects with 3 rep ications available). Accuracy of miR-34b-3p varies according to cut-point and number of replications below the cut-point (AUC was 0.68 with a outpoint for positivity of 39), with sensitivity reaching 36% with a specificity of 100%.

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Claims

1 ) Method for in vitro diagnosis of Amyotrophic Lateral Sclerosis, said method comprising the detection, in a biological sample, of all, two or at least one of the following exosomal RNAs: miR-34b-3p, SOD2, SLC25A21 , wherein said RNAs are expressed in Amyotrophic Lateral Sclerosis.

2) Method according to claim 1 , which comprises:

a) extracting exosomes in a biological sample;

b) extracting RNAs from exosomes;

c) detecting all, two or at least one of the following exosomal RNAs: miR-34b-3p, SOD2, SLC25A21 , wherein said RNAs are present in Amyotrophic Lateral Sclerosis.

3) Method according to anyone of claims 1 -2, said method further comprising the detection, in a biological sample, of all, more than one or one of the following exosomal mirRNAs: miR-335-5p, miR-99b-5p, miR- 136-5p, miR-130b-3p, miR-326,

wherein said miR-335-5p, miR-99b-5p, miR-136-5p, miR-130b-3p, miR-326 are expressed in healthy subjects.

4) Method according to anyone of claims 1 -3, said method further comprising the detection, in a biological sample, of all, more than one or one of the following exosomal mirRNAs: miR-124-3p, miR-222-3p, miR- 143-3p, let-7b-5p, let-7d-3p, miR-145-5p, let-7i-5p, miR-9-3p, miR-15a-5p, let-7a-5p,

wherein miR-124-3p, miR-222-3p, miR-143-3p, let-7b-5p, let-7d-3p, miR-145-5p, let-7i-5p, miR-9-3p, miR-15a-5p are downregulated in Amyotrophic Lateral Sclerosis in comparison with healthy controls, whereas let-7a-5p is upregulated in Amyotrophic Lateral Sclerosis in comparison with healthy controls.

5) Method according to anyone of claims 1 -4, said method further comprising the detection, in a biological sample, of all, more than one or one of the following exosomal mRNAs: SLC25A3, CPT1 B, OPA1 , SLC25A16 and SLC25A24, wherein SLC25A3 and CPU B are upregulated in Amyotrophic Lateral Sclerosis in comparison with healthy controls, whereas OPA1 , SLC25A16 and SLC25A24 are downregulated in Amyotrophic Lateral Sclerosis in comparison with healthy controls.

6) Method according to any one of claims 1 -5, wherein the biological sample is a liquid biological sample such as blood, nasal lavage, supernatant of cells extracted from biological tissues.

7) Method according to any one of claims 1 -6, wherein said method is carried out by Real Time PCR, Droplet Digital PCR, Microarray, RNA Hybridization Methods such as Northern Blot or Dot Blot, RNA Next Generation Sequencing.

8) Use of all, more than one or at least one of the following RNAs as biomarkers for the in vitro diagnosis of Amyotrophic Lateral Sclerosis: miR- 34b-3p, SOD2, SLC25A21 , miR-335-5p, miR-99b-5p, miR-136-5p, miR- 130b-3p, miR-326, miR-124-3p, miR-222-3p, miR-143-3p, let-7b-5p, let- 7d-3p, miR-145-5p, let-7i-5p, miR-9-3p, miR-15a-5p, let-7a-5p, SLC25A3, CPU B, OPA1 , SLC25A16 or SLC25A24.