WO2019139901A1 - Nasal exosomes for non-invasive sampling of cns proteins - Google Patents

Abstract

Methods and devices for isolating and using cerebrospinal fluid (CSF)-derived olfactory mucus exosomes (CDOMEs), e.g., for the non-invasive detection of neurodegenerative disease.

Description

NASAL EXQSQMES FOR NQN-INVASIVE SAMPLING OF

CNS PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 62/616,882, filed on January 12, 2018. The entire contents of the foregoing are incorporated herein by reference.

TECHNICAL FIELD

Described herein are methods and devices for isolating and using

cerebrospinal fluid (CSF)-derived olfactory mucus exosomes (CDOMEs), e.g., for the non-invasive detection of neurodegenerative disease.

BACKGROUND

Alzheimer’s Disease(AD) represents the most prevalent neurodegenerative disease; the World Health Organization currently estimates that approximately 35.6 million people are afflicted by AD worldwide. In the United States, approximately 7 million people over 65 suffer from AD and this number is expected to triple by 2050. According to the 2013 facts and figures from the Alzheimer’s Association, although the number of deaths from major diseases such as cancer and cardiovascular disease has declined in the past decade, the number of deaths related to AD has increased 68% during the 2000 to 2010 period. Major advances in treatment for the various diseases, except AD, are reflected in these statistics, as well as the increasing longevity of our population. The social and psychological burden associated with caring for patients with AD is difficult to quantify. The health-care costs associated with managing these individuals however are profound. In the United States alone, AD related health-care costs were estimated in 2010 to surpass $170 billion and projected to exceed $1 trillion by 2050[l]

SUMMARY

Provided herein are methods and devices for detecting a neurological disease in a subject based on the detection of biomarkers in CSF Derived Olfactory Mucus Exosomes (CDOMEs) from the subject. Thus, described herein are methods that include isolating CSF Derived Olfactory Mucus Exosomes (CDOMEs) from the subject; determining a level of a biomarker associated with a neurological disease in the CDOMEs; and diagnosing a neurological disease in the subject based on the level of the biomarker associated with the neurological disease in the CDOMEs from the subject.

Also described herein are methods for diagnosing Alzheimer’s Disease (AD) in a subject that include isolating CSF Derived Olfactory Mucus Exosomes

(CDOMEs) from the subject; determining a level of a biomarker associated with AD in the CDOMEs; and

diagnosing AD in the subject based on the level of the biomarker associated with the neurological disease in the CDOMEs from the subject.

In some embodiments, the methods include comparing the level of the biomarker in the CDOMEs to a reference level, and diagnosing the disease when the level of the biomarker in the CDOMEs is above the reference level.

In some embodiments, the marker is selected from the group consisting of Ab

1-42, Ab 1-42/Ab 1-40 ratio, P-S396-X, P-T181-X, and total x.

In some embodiments, the methods include selecting and optionally administering a treatment for the neurological disease to the subject.

In some embodiments, the methods include selecting and optionally administering a treatment for AD to the subject.

Also provided herein are methods for monitoring the efficacy of a treatment for a neurological disease, e.g., Alzheimer’s Disease (AD), in a subject. The methods can include isolating a first sample comprising CSF Derived Olfactory Mucus Exosomes (CDOMEs) from the subject; determining a level of a biomarker associated with the neurological disease, e.g., AD, in the first sample; treating the subject;

isolating a second sample comprising CSF Derived Olfactory Mucus Exosomes (CDOMEs) from the subject; determining a level of the biomarker associated with neurological disease, e.g., AD, in the second sample; and identifying the treatment as successful in a subject when the level of the biomarker in the second sample is below the level of the biomarker in the first sample.

Neurological diseases that can be diagnosed and monitored within the present methods include AD, PD, HD, psychiatric disorders, cancer (e.g., brain or CNS cancer), and traumatic brain injury. Further, provided herein are nasal cavity sampling devices for use in the present methods that include (i) a nasal insert sized and shaped to be inserted into a nasal opening of a nasal cavity of a patient, the nasal insert being configured to receive a first flow of medical fluid from a syringe, the nasal insert comprising: a first aperture arranged at a distal end of the nasal insert and configured to deliver a medical fluid along a trajectory towards an olfactory region in the nasal cavity of the patient, and a second aperture arranged proximal to the first aperture and configured to receive a second flow of medical fluid from the nasal cavity of the patient; and (ii) a collection container fluid coupled to the nasal insert and configured to receive the second flow of the medical fluid from the second aperture of the nasal insert.

In some embodiments, the nasal cavity sampling device includes a syringe fluidly coupled to the nasal insert and configured to deliver the first flow of the medical fluid to the first aperture of the nasal insert, the syringe containing the medical fluid. In some embodiments, the medical fluid is saline. In some

embodiments, the collection container contains a preservative, e.g., a protease inhibitor or a nuclease inhibitor.

In some embodiments, the collection container comprises a tube extending from the collection container and arranged to be disposed in the patient’s mouth in order for the patient to apply suction to the collection container, the suction drawing the medical fluid from the second aperture and into the collection container.

In some embodiments, the tube comprises a valve configured to maintain a negative pressure in the collection container after the patient’s application of suction.

In some embodiments, the collection container comprises a level configured to aid the patient in positioning the collection container at a given level with respect to the nasal insert.

In some embodiments, the nasal insert comprises a conical surface sized and positioned to expose the first aperture to the olfactory region when the nasal insert is disposed in the nasal opening, the conical surface including the first and second apertures.

In some embodiments, the conical surface is sized and positioned to dispose the second aperture along a nasal floor of the nasal cavity when the nasal insert is disposed in the nasal opening. In some embodiments, the conical surface is sized and positioned to sealingly interface with the nasal opening of the patient when the nasal insert is disposed in the nasal opening.

Also provided herein are kits for use in the present methods. The kits can include one or more of: a sponge for sample collection; a delivery device for placing the sponge; a collection container; a protease inhibitor and/or RNAse inhibitor for sample preservation; sterile saline; and a cold or freezer pack for specimen preservation during shipment.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. DESCRIPTION OF DRAWINGS

FIG. 1 is an illustration of the anatomy of the human olfactory mucosa (black box in right figure) demonstrating the direct connection between the olfactory mucosa in the brain via the olfactory nerves. The black arrows demonstrate the diffusion pathway for CDOMEs from the CSF space, through the cribriform plate, and into the olfactory mucus where they can be non-invasively collected. Within the nose,

CDOMEs are swept into the nasopharynx (N) by mucociliary action where they are diluted by secreted sinonasal exosomes (white arrows) before being swallowed.

Figures 2A-B are Transmission electron microscopy (TEM) images demonstrating (A) negative control whole mounted exosomes purified from nasal lavage (bar = 100 nm) confirming the typical exosome size and morphology and (B) immunogold labeling of exosome marker CD63 localizing to the exosome membrane.

Figure 2C. Histogram demonstrating the presence of B2-transferrin, a protein only found in CSF, perilymph, and aqueous humor, in CDOMEs. Figure 2D. Histogram demonstrating the presence of p-tau S396, total tau, and AB 1-42 in CDOMEs.

Figures 3A-B are schematic illustrations of an exemplary device and method for isolating CDOMEs. DETAILED DESCRIPTION

Symptoms of AD include loss of memory and impairment of cognitive function but it is often difficult to diagnose, especially in the early clinical stages. This has dramatically limited the development of new therapies for AD as patient selection requires the presence of symptoms. By the time symptoms emerge, there has already been a significant loss of functional brain which profoundly reduces the efficacy of any potential therapy.

One of the most significant barriers to the development of new treatments for AD is a lack of non-invasive biomarkers to enable preclinical diagnosis, monitor disease progression, and quantify response to therapy. A biomarker is an indicator of the presence or extent of a biological process that is directly linked to the clinical manifestations and outcome of a particular disease. The development of a low cost, non-invasive, yet highly accurate strategy for biomarker detection represents a major unmet need in the management of AD. Such a method would enable serial, longitudinal, screening for AD which would not only facilitate the diagnosis at an earlier stage where neuroprotective treatment would be more effective, but would also catalyze the development of new therapies whose efficacy could be more reliably tracked over time.

Currently the most successful biomarker in AD requires sampling of the cerebrospinal fluid (CSF) for amyloid beta(AB) and tau (x) proteins. Sampling CSF through a lumbar puncture is currently the least invasive direct method for assessing pathologic alterations occurring within the CNS. Not only bathing the superficial portions of the brain, spinal cord, and portions of the cranial and spinal nerves, CSF also communicates directly with the cerebral ventricles and extracellular fluid of these structures. As such, many believe that CSF provides an optimal source of various biomarkers for diverse pathobiologic events occurring within the CNS, including AD

[4] The most consistent AD correlates within CSF have been related to

concentrations of a proteolytic fragment of the amyloid precursor protein, Ab 1-42, in addition to total tau (t-t) and p-x levels[5][6]. From additional CSF analyses, an“AD signature” has been proposed, featuring low levels of Ab (Ab 1-42 or Ab 1-42/ Ab 1- 40 ratio) and elevated quantities of t-t and p-x [5] [6] [7] Although the sensitivity of CSF measurements of AB42 and total x alone are below 60%, the combination of both of these markers together is reported to have a sensitivity of around 90% in

Alzheimer’s disease diagnosis, which in combination with clinical evaluation and imaging ensures high accuracy of the clinical diagnosis [2] Differences in tau phosphorylation epitopes have also been used to define stages of neurofibrillary tangle(NFT) development within the CNS in AD [7] [8] Specifically, elevated levels of p- x S262 (serine 262) or p- x T181 (threonine 181) are noted in earlier stages of NFT development, whereas p-tau S396 accumulates in later Braak stages when x accumulations are extracellular [7] Times for progression from preclinical stages to clinically apparent AD with threshold detectable amyloid deposition and abnormal elevation of CSF p- x proteins are estimated to be up to 17 years [9] The potential prognostic sensitivity of protein biomarkers is supported by the timing of induction of AD-like disease in rodent models after the transgenic overexpression of putatively neuropathogenic proteins [10] The sensitivity of these AD biomarkers to both preclinical and clinical stages of AD demonstrate the potential benefits of routine biomarker analysis.

However, this technique is invasive, expensive, and has been associated with significant complications. CSF sampling for AD biomarker analysis can be associated with significant risks. These include spinal headache, local back or radiating leg pains, meningitis, epidural abscess, subdural hematoma, and even death [11] [12] [13]. Additionally, low CSF pressure/volume states [14][15], are prevalent in the elderly which can predispose these subjects to unsuccessful taps[l6] and a higher risk of other sequelae. Furthermore, sampling requires specialized training and the use of a sterile technique to minimize morbidity associated with accessing lumbar CSF [17] Despite the potential significant benefits of serial CSF correlations within longitudinal preclinical to clinical AD studies, the risks and costs associated with lumbar puncture are far too great to justify routine sampling in asymptomatic patients.

The increasing pressure to develop accurate biomarkers of preclinical AD coupled with the profound scale of the disease has led many to examine the costs and benefits associated with the various biosignatures. Ultimately, these analyses will be relevant to the overall cost of care for AD. The costs are not insignificant for accessing various biomarkers. Based on available information, calculated costs for CSF collection for biomarker analysis range from $350,000 to >$1,000,000 per 1000 subjects and are even greater for neuroimaging studies. These costs are an order of magnitude greater than those associated with other peripheral sampling methods such as blood draws. These substantial costs suggest that biomarker modalities applicable to screening large populations of preclinical subjects require low costs and high accuracy. The systematic application of these methods will be required to promote the development and validation of disease-modifying therapeutics [3]

Exosomes are a class of endosome-derived membrane vesicles shed by neural cells, that contain proteins and other constituents of their cellular origin [18]

Exosomes are 30-150 nm vesicles, surrounded by a lipid bilayer, that have a density of 1.13-1.19 g/ml. Exosomes have been detected in a wide range of body fluids including blood, lymph, CSF, and urine [1] [2] Biophysically, exosomes are equivalent to cytoplasm enclosed in a lipid bilayer with the external domains of transmembrane proteins exposed to the extracellular environment [3] The biogenesis of exosomes is controlled by the endosomal sorting complex required for transport. These events lead to the development late endosome/multivesicular bodies which can then be recycled back into the plasma membrane and released as exosomes. This process leads to exosomes becoming strongly enriched in markers including the tetraspanins CD63, CD9, CD81, and CD82 which can be used to detect their presence and quantity [1] Exosomes are capable of transporting a wide range of cargo including growth factors and their receptors, DNA, mRNA, and microRNA. Further studies have demonstrated that exosomes are able to shuttle this cargo, including integral membrane proteins such as the chemokine receptor CCR5 [4], to adjacent cells.

CSF derived exosomes have been previously shown to express specific microRNA profiles in both Parkinson’s and Alzheimer’s disease [see, e.g., reference 19] Furthermore, exosomes accept amyloid precursor protein from early endosomes, after its cleavage by B-secretase. and the Ab peptide fragments subsequently generated by g-secretase are secreted in exosomes [20] Although this exosome pathway accounts for only a small portion of the total Ab peptides in neural plaques, it constitutes a prionoid-like mechanism for the CNS spread of proteinopathies [21] The detection of exosome signature proteins in neural amyloid plaques supports the possibility of their role in the generation of AD associated lesions [20] CSF derived exosomes are capable of crossing the blood-brain barrier and have been detected in a variety of peripheral body fluids including blood and urine [22] [23] For example, Fiandaca et al. [22] demonstrated that total tau, P-Tl8l-tau, and Ab 1-42 were detectable in blood-derived serum exosomes of neural origin and were elevated in patients with AD relative to control exosomes. While CSF-derived exosome collection from peripheral body fluids represents a promising a reservoir for AD biomarkers, their utility is limited by dilution and contamination by other blood and organ-derived exosomes as well as progressive degradation by plasma proteases and nucleases during circulation.

The nasal lining contains a region, known as the olfactory mucosa, which has a direct communication pathway with the CSF space. As olfactory neurons, which are responsible for the sense of smell, penetrate into the olfactory mucosa they are surrounding by a sheath of cerebrospinal fluid (Figure 1). Consequently, up to 30% of CSF is resorbed through the nasal mucosa [24] As exosomes are capable of crossing the BBB [22] [23], as shown herein, CSF derived exosomes are able to diffuse into the olfactory mucosa and can be detected in olfactory mucus. Furthermore, these CSF derived olfactory mucus exosomes (CDOMEs) can be recovered for sensitive quantification and protein expression analysis (Figures 2A-D).

Taken together, the present findings suggest that CDOMEs represent a novel and superior reservoir for CNS-derived biomarker sampling that has the potential to revolutionize both the diagnosis and treatment of neurological diseases such as AD. This technique therefore has the potential to overcome all of the previously discussed drawbacks of current CNS-derived biomarker sampling techniques. It is completely non-invasive and can therefore be performed serially over time in an outpatient or even home based setting with minimal cost relative to lumbar puncture. Furthermore, as this method directly detects CSF derived exosomes, it has the potential to significantly improve diagnostic accuracy over other peripheral sampling techniques. Finally, the safety profile of this method provides the possibility to realize the dream of longitudinal screening of large pre-clinical and clinical AD patient populations which could, in turn, lead to the discovery of both new and more sensitive biomarkers as well as novel AD therapies which can be utilized at ever earlier stages of the disease. Methods of Diagnosis

Included herein are methods for diagnosing a neurological disease in a subject, e.g., Alzheimer’s Disease, brain cancer, brain trauma, and psychiatric disorders. The methods rely on detection of a biological marker or a plurality of biological makers of a particular disease state or disease susceptibility in CDOMEs. Biological markers commonly used include polypeptides or nucleic acids, including but not restricted to proteins, antibodies, DNA, RNA, miRNA, and lncRNA, that are characteristic of a particular disease state or disease susceptibility. The methods include obtaining a sample comprising CDOMEs from a subject, and evaluating the presence and/or level of a biomarker related to a neurological disease in the sample, and comparing the presence and/or level of the biomarker related to a neurological disease with one or more references, e.g., a control reference that represents a normal level of the biomarker, e.g., a level in an unaffected subject, and/or a disease reference that represents a level of the proteins associated with a neurological disease, e.g., a level in a subj ect having AD .

Samples and Isolation of CSF derived olfactory mucus exosomes (CDOMEs)

As used herein the term“sample”, when referring to the material to be tested for the presence of a biological marker using the method of the invention, means a sample comprising CDOMEs. The CDOMEs can be easily sampled and isolated using a sponge or other absorbent device capable of absorbing mucus placed in the olfactory cleft. An exemplary sponge is a compressed sterile 2x2x5mm or 2x3x15mm poly vinyl-alcohol sponge (e.g., commercially available from Medtronics, and designed to be used in the nose for hemostasis and stenting after sinus surgery or in the setting of nose bleeds). A high volume diluted strategy can also be used, e.g., a completely non- invasive saline lavage that includes the use of a saline lavage directed at the olfactory cleft, e.g., a lavage of at least 50 pL up to about 30 mL, e.g., 50 pL - 10 mL, 50 pL - 30 mL, 10-30 mL, e.g., 18-25 mL, e.g., 20 mL. The lavage can be completed and collected, e.g., using a device as shown in Figure 3.

The sponge or lavage can be stored, e.g., at -80°C, preferably in the presence of a biomarker preservative, e.g., a protease inhibitor or nuclease inhibitor (such as RNase inactivating enzymes) until isolation.

A number of methods can be used to isolate CDOMEs from the sample, e.g., centrifugation (e.g., traditional ultracentrifugation (UCF) as described in [25]); chromatography; filtration; polymer-based precipitation; and immunological separation methods; see Yakimchik, Exosomes: isolation and characterization methods and specific markers, 2016-11-30, dx.doi.org/l0. l3070/mm.en.5. l450, and references cited therein. An exemplary polymer based exosome precipitation system is the ExoQuick from System Biosciences. In an exemplary UCF method, mucus and irrigant samples can be diluted, e.g., in 150 pL of 1 x phosphate buffered saline (PBS) with Protease Inhibitor Cocktail. Cellular debris can be pelleted by centrifugation, e.g., at 45 min at 12,000 x g at 4°C. The supernatant can then be suspended in PBS, e.g., 4.5mL of PBS in polypropylene tubes, and ultracentrifuged, e.g., for 2 hours at 110,000 x g, at 4°C. The supernatant can then be collected and the pellet resuspended in PBS, e.g., in 4.5 mL lx PBS. The suspension can be filtered, e.g., through a 0.22- pm filter, and collected in a fresh tube. The filtered suspension can then be centrifuged again, e.g., for 70 min at 110,000 x g at 4°C. The supernatant can then be collected and the pellet resuspended in a buffer, e.g., in PBS, e.g., in 200 pl PBS with protease inhibitor.

Various methods known within the art can be used for the identification and/or isolation and/or purification of a biological marker from a sample. An“isolated” or “purified” biological marker is substantially free of cellular material or other contaminants from the cell or tissue source from which the biological marker is derived i.e. partially or completely altered or removed from the natural state through human intervention. For example, nucleic acids contained in the sample can be first isolated according to standard methods, for example using lytic enzymes, chemical solutions, or isolated by nucleic acid-binding resins following the manufacturer’s instructions. Exosomal Biomarkers in Neurological Disease

A number of biomarkers have been described in the art that can be used in the present methods to detect and monitor neurological disease. For example, in patients having or suspected of having AD, amyloid beta (Ab) and tau (x) proteins can be analysed, e.g., Ab 1-42 or Ab 1-42/ Ab 1-40 ratio, and/or t-t and p-x; a-synuclein and phosphorylated tau in Parkinson’s disease (PD) (Hall et al, Neurology. (2015) 84:57- 63); and ubiquitin, prothrombin, haptoglobin and Apolipoprotein A-IV (ApoA) in Huntington’s Disease (HD) (Byrne and Wild, J. Huntington's Disease 5 (1): 1-13 (2016)). Differences in tau phosphorylation epitopes have also been used to define stages of neurofibrillary tangle (NFT) development within the CNS in AD [10] [12] Specifically, elevated levels of p-S262 (serine 262)-tau or r-T181 (threonine l8l)-tau are noted in earlier stages of NFT development, whereas p-tau S396 accumulates in later Braak stages when tau accumulations are extracellular [10] See also van Waalwijk van Doom et al, J Alzheimers Dis. 2017 Dec 3. doi: 10.3233/JAD-160668.

Paterson et al. [42] identified five biomarkers in CSF that differentiate neurochemical AD from non- AD in two independent clinical populations from different centers. After adjustment for multiple comparisons, five proteins were elevated significantly in AD CSF compared with non-AD CSF in both cohorts: malate dehydrogenase; total APOE; chitinase-3-like protein 1 (YKL-40); osteopontin and cystatin C. These markers can also be used in the present methods.

Other neurologic disorders that can be diagnosed by detecting markers present in CDOMEs as described herein include psychiatric and oncologic disorders as well as traumatic brain injury (TBI).

For example, 5-hydroxyindoleacetic acid (5-HIAA) levels in CSF have been linked to depression (Boksa et al,. J Psychiatry Neurosci 20l3;38(2):75-7).

TBI has been linked to elevations in tau protein (see, e.g., Bulut et al, Adv Ther. 2006 Jan-Feb;23(l): 12-22; Acosta et al, J Cell Physiol. 2017 Mar;232(3):665- 677; Gerson et al, J. Neurotrauma. November 2016, 33(22): 2034-2043.

For diagnosing cancer, CSF levels of carcinoembryonic antigen (CEA) have been shown to be increased in several human brain malignances (see, e.g., Batabyal et al, Neoplasma 2003;50:377-9; Moldrich et al, Acta Neurol Belg 2010;110:314-20; Nakagawa et al, J Neurooncol 1992;12: 111-20; Wang et al, Cancer Biomark 2013;13: 123-30). Other proteins present in CSF and thus expected to be useful as biomarkers in the present methods include MMP-2, MMP-9, and MMP-9/NGAL, Apolipoprotein A-II, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), Prostaglandin D2 synthase, insulin-like growth factor (IGF), Insulin-like growth factor-binding protein-3 (IGFBP-3), IGFBP-2, Polysialic-neural cell adhesion molecule (PSA-NCAM), apolipoproteins E and J, Osteopontin, Tau, Metallothionein and others described in Russell et al,. Front Pediatr. 2013; 1 : 7; Kim et al, Med Sci Monit 20l l;l8:BR450-60 (meningioma); Okamoto et al, Cancer Res 2006;66: 10199-204 (meningioma); Vankalakunti et al, Neuropathology 2007;27:407- 12 (MIB-l in meningioma); Wulfkuhle et al., Adv Exp Med Biol 2003;532:59-68; Kalinina et al, Neuro Oncol 2011;13:926-42 (glioma); Gollapalli et al., Proteomics 2012;12:2378-90 (glioblastoma multiforme); Iwadate et al, Cancer Res

2004;64:2496-501 (glioma); Khwaja et al, Proteomics 2006;6:6277-87; Samuel et al, J Neurooncol 2014;118:225-38; Qaddoumi et al., Childs Nerv Syst 2012;28:1017-24 (intracranial germ cell tumors); Nishizaki et al., J Clin Neurosci 2001;8:27-30

(intracranial germ cell tumors); Watanabe et al., Pediatr Neurosurg 2012;48: 141-5 (placental alkaline phosphatase in intracranial germinomas); Miyanohara et al, J Neurosurg 2002;97: 177-83 (soluble c-kit in germ cell tumors); Khwaja et al, J Proteome Res 2007;6:559-70 (astrocytoma); and Khwaja et al, Clin Cancer Res 2006;12:6331-6 (Attractin in astrocytoma). See Shalaby et al, Targeting

cerebrospinal fluid for discovery of brain cancer biomarkers. J Cancer Metastasis Treat 2016;2: 176-87.

The presence and/or level of a protein can be evaluated using methods known in the art, e.g., using standard electrophoretic and quantitative immunoassay methods for proteins, including but not limited to, Western blot; enzyme linked

immunosorbent assay (ELISA); biotin/avidin type assays; protein array detection; radio-immunoassay; immunohistochemistry (IHC); immune-precipitation assay; FACS (fluorescent activated cell sorting); mass spectrometry (Kim (2010) Am J Clin Pathol 134: 157-162; Yasun (2012) Anal Chem 84(l4):6008-60l5; Brody (2010) Expert Rev Mol Diagn 10(8): 1013-1022; Philips (2014) PLOS One 9(3):e90226;

Pfaffe (2011) Clin Chem 57(5): 675-687). The methods typically include revealing labels such as fluorescent, chemiluminescent, radioactive, and enzymatic or dye molecules that provide a signal either directly or indirectly. As used herein, the term “label” refers to the coupling (i.e. physically linkage) of a detectable substance, such as a radioactive agent or fluorophore (e.g. phycoerythrin (PE) or indocyanine (Cy5), to an antibody or probe, as well as indirect labeling of the probe or antibody (e.g. horseradish peroxidase, HRP) by reactivity with a detectable substance.

In some embodiments, an ELISA method may be used, wherein the wells of a mictrotiter plate are coated with an antibody against which the protein is to be tested. The sample containing or suspected of containing the biological marker is then applied to the wells. After a sufficient amount of time, during which antibody-antigen complexes would have formed, the plate is washed to remove any unbound moieties, and a detectably labelled molecule is added. Again, after a sufficient period of incubation, the plate is washed to remove any excess, unbound molecules, and the presence of the labeled molecule is determined using methods known in the art. Variations of the ELISA method, such as the competitive ELISA or competition assay, and sandwich ELISA, may also be used, as these are well-known to those skilled in the art. Next generation ultrasentive ELISA can also be used, e.g., a single molecule array (SIMOA) digital ELISA may also be used. For example, for each assay, capture antibody is first covalently conjugated to magnetic particles utilizing a standard EDC coupling procedure and detection antibody was biotinylated. In the first step of the assay, antibody coated paramagnetic capture beads, biotinylated detection antibodies, and samples are combined, during which target molecules present in the sample are captured by the capture beads and labeled with the biotinylated detection antibodies. After washing, a conjugate of streptavidin^-galactosidase (NbO) is mixed with the capture beads where S G bound to the biotin, resulting in enzyme labeling of captured target molecules. Following a second wash, the capture beads are resuspended in a resorufm b-D-galactopyranoside (RGP) substrate solution and transferred to the Simoa array disc for detection.

In some embodiments, an IHC method may be used. IHC provides a method of detecting a biological marker in situ. The presence and exact cellular location of the biological marker can be detected. Typically, a sample is fixed with formalin or paraformaldehyde, embedded in paraffin, and cut into sections for staining and subsequent inspection by confocal microscopy. Current methods of IHC use either direct or indirect labelling. The sample may also be inspected by fluorescent microscopy when immunofluorescence (IF) is performed, as a variation to IHC.

Mass spectrometry, and particularly matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS), is useful for the detection of biomarkers of this invention. (See U.S. Patent No. 5,118,937; 5,045,694; 5,719,060; 6,225,047).

In some embodiments, Slow Off-rate Modified Aptamer (SOMAmer)-based capture array called‘SOMAscan’ (SomaLogic, Inc, Boulder, Colorado) can be used. This approach uses chemically modified nucleotides to transform a protein signal to a nucleotide signal that can be quantified using relative florescence on microarrays.

This assay has been shown to have a median intra- and inter-run coefficient of variation of ~5%. The presence and/or level of a nucleic acid can be evaluated using methods known in the art, e.g., using polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative or semi-quantitative real-time RT- PCR, digital PCR i.e. BEAMing ((Beads, Emulsion, Amplification, Magnetics) Diehl (2006) Nat Methods 3:551-559) ; RNAse protection assay; Northern blot; various types of nucleic acid sequencing (Sanger, pyrosequencing, NextGeneration

Sequencing); fluorescent in-situ hybridization (FISH); or gene array/chips)

(Lehninger Biochemistry (Worth Publishers, Inc., current addition; Sambrook, et al, Molecular Cloning: A Laboratory Manual (3. Sup.rd Edition, 2001); Bernard (2002) Clin Chem 48(8): 1178-1185; Miranda (2010) Kidney International 78: 191-199;

Bianchi (2011) EMBO Mol Med 3:495-503; Taylor (2013) Front. Genet. 4: 142; Yang (2014) PLOS One 9(1 l):el 10641); Nordstrom (2000) Biotechnol. Appl. Biochem.

31(2): 107-112; Ahmadian (2000) Anal Biochem 280: 103-110. In some

embodiments, high throughput methods, e.g., protein or gene chips as are known in the art (see, e.g., Ch. 12, Genomics, in Griffiths et al., Eds. Modem genetic Analysis, 1999, W. H. Freeman and Company; Ekins and Chu, Trends in Biotechnology, 1999, 17:217-218; MacBeath and Schreiber, Science 2000, 289(5485): l760-l763; Simpson, Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 2002; Hardiman. Microarrays Methods and Applications: Nuts & Bolts, DNA Press, 2003), can be used to detect the presence and/or level of a biomarker.

Measurement of the level of a biomarker can be direct or indirect. For example, the abundance levels of biomarkers can be directly quantitated. Alternatively, the amount of a biomarker can be determined indirectly by measuring abundance levels of cDNA, amplified RNAs or DNAs, or by measuring quantities or activities of RNAs, or other molecules that are indicative of the expression level of the biomarker. In some embodiments a technique suitable for the detection of alterations in the structure or sequence of nucleic acids, such as the presence of deletions, amplifications, or substitutions, can be used for the detection of biomarkers of this invention.

RT-PCR can be used to determine the expression profiles of biomarkers (U.S. Patent No. 2005/0048542A1). The first step in expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction (Ausubel et al (1997) Current Protocols of Molecular Biology, John Wiley and Sons). To minimize errors and the effects of sample-to- sample variation, RT-PCR is usually performed using an internal standard, which is expressed at constant level among tissues, and is unaffected by the experimental treatment. Housekeeping genes are most commonly used.

Gene arrays are prepared by selecting probes which comprise a polynucleotide sequence, and then immobilizing such probes to a solid support or surface. For example, the probes may comprise DNA sequences, RNA sequences, co-polymer sequences of DNA and RNA, DNA and/or RNA analogues, or combinations thereof. The probe sequences can be synthesized either enzymatically in vivo, enzymatically in vitro (e.g. by PCR), or non-enzymatically in vitro. Comparison to a Reference Level

In some embodiments, the presence and/or level of a biomarker is comparable to the presence and/or level of the protein(s) in the disease reference, and the subject has one or more symptoms associated with a neurological disease, then the subject has the neurological disease. In some embodiments, the subject has no overt signs or symptoms of neurological disease, but the presence and/or level of one or more of the proteins evaluated is comparable to the presence and/or level of the protein(s) in the disease reference, then the subject has an increased risk of developing the

neurological disease or has an early stage of the disease. In some embodiments, once it has been determined that a person has the neurological disease, or has an increased risk of developing neurological disease, then a treatment, e.g., as known in the art or as described herein, can be administered.

Suitable reference values can be determined using methods known in the art, e.g., using standard clinical trial methodology and statistical analysis. The reference values can have any relevant form. In some cases, the reference comprises a predetermined value for a meaningful level of a biomarker, e.g., a control reference level that represents a normal level of the biomarker, e.g., a level in an unaffected subject or a subject who is not at risk of developing a disease described herein, and/or a disease reference that represents a level of the proteins associated with conditions associated with a neurological disease, e.g., a level in a subject having a neurological disease (e.g., AD).

The predetermined level can be a single cut-off (threshold) value, such as a median or mean, or a level that defines the boundaries of an upper or lower quartile, tertile, or other segment of a clinical trial population that is determined to be statistically different from the other segments. It can be a range of cut-off (or threshold) values, such as a confidence interval. It can be established based upon comparative groups, such as where association with risk of developing disease or presence of disease in one defined group is a fold higher, or lower, (e.g.,

approximately 2-fold, 4-fold, 8-fold, 16-fold or more) than the risk or presence of disease in another defined group. It can be a range, for example, where a population of subjects (e.g., control subjects) is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group, or into quartiles, the lowest quartile being subjects with the lowest risk and the highest quartile being subjects with the highest risk, or into n-quantiles (i.e., n regularly spaced intervals) the lowest of the n-quantiles being subjects with the lowest risk and the highest of the n- quantiles being subjects with the highest risk.

In some embodiments, the predetermined level is a level or occurrence in the same subject, e.g., at a different time point, e.g., an earlier time point.

Subjects associated with predetermined values are typically referred to as reference subjects. For example, in some embodiments, a control reference subject does not have a neurological disorder described herein (e.g. AD).

A disease reference subject is one who has (or has an increased risk of developing) a neurological disease. An increased risk is defined as a risk above the risk of subjects in the general population.

Thus, in some cases the level of a biomarker in a subject being less than or equal to a reference level of the biomarker is indicative of a clinical status (e.g., indicative of a disorder as described herein, e.g., AD. In other cases, the level of the biomarker in a subject being greater than or equal to the reference level of the biomarker is indicative of the absence of disease or normal risk of the disease. In some embodiments, the amount by which the level in the subject is the less than the reference level is sufficient to distinguish a subject from a control subject, and optionally is a statistically significantly less than the level in a control subject. In cases where the level of the biomarker in a subject being equal to the reference level of the biomarker, the“being equal” refers to being approximately equal (e.g., not statistically different).

The predetermined value can depend upon the particular population of subjects (e.g., human subjects) selected. For example, an apparently healthy population will have a different‘normal’ range of levels of the biomarker than will a population of subjects which have, are likely to have, or are at greater risk to have, a disorder described herein. Accordingly, the predetermined values selected may take into account the category (e.g., sex, age, health, risk, presence of other diseases) in which a subject (e.g., human subject) falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.

In characterizing likelihood, or risk, numerous predetermined values can be established. Methods of T reatment

The methods described herein can be used for the selection of patients for treatment, e.g., treatments that impact senile plaques, reduce amyloid beta and tau, reduce neuroinflammation, restore neuronal function, and restore brain rhythms including gamma rhythms that are known to play a role in AD. The methods can also be used to determine whether a treatment being tested in a clinical trial is effective.

In some embodiments, the neurological disorder is a neurodegenerative disorder. Neurodegenerative disorders are a class of neurological diseases that are characterized by the progressive loss of the structure and function of neurons and neuronal cell death. Inflammation has been implicated for a role in several neurodegenerative disorders. Progressive loss of motor and sensory neurons and the ability of the mind to refer sensory information to an external object is affected in different kinds of neurodegenerative disorders. Non-limiting examples of neurodegenerative disorders include Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS, e.g., familial ALS and sporadic ALS), and multiple sclerosis (MS).

A health care professional may diagnose a subject as having a

neurodegenerative disorder by the assessment of one or more symptoms of a neurodegenerative disorder in the subject. Non-limiting symptoms of a

neurodegenerative disorder in a subject include difficulty lifting the front part of the foot and toes; weakness in arms, legs, feet, or ankles; hand weakness or clumsiness; slurring of speech; difficulty swallowing; muscle cramps; twitching in arms, shoulders, and tongue; difficulty chewing; difficulty breathing; muscle paralysis; partial or complete loss of vision; double vision; tingling or pain in parts of body; electric shock sensations that occur with head movements; tremor; unsteady gait; fatigue; dizziness; loss of memory; disorientation; misinterpretation of spatial relationships; difficulty reading or writing; difficulty concentrating and thinking; difficulty making judgments and decisions; difficulty planning and performing familiar tasks; depression; anxiety; social withdrawal; mood swings; irritability; aggressiveness; changes in sleeping habits; wandering; dementia; loss of automatic movements; impaired posture and balance; rigid muscles; bradykinesia; slow or abnormal eye movements; involuntary jerking or writhing movements (chorea); involuntary, sustained contracture of muscles (dystonia); lack of flexibility; lack of impulse control; and changes in appetite. A health care professional may also base a diagnosis, in part, on the subject’s family history of a neurodegenerative disorder. A health care professional may diagnose a subject as having a neurodegenerative disorder upon presentation of a subject to a health care facility (e.g., a clinic or a hospital). In some instances, a health care professional may diagnose a subject as having a neurodegenerative disorder while the subject is admitted in an assisted care facility. Typically, a physician diagnoses a neurodegenerative disorder in a subject after the presentation of one or more symptoms.

CDOME Sampling Devices

FIG. 3A is an illustration of a sampling device 300 obtaining a sample 31 from the nasal cavity 302 of a patient’s head 301. The sampling device 300 includes a syringe 320, a nasal insert 310, a collection container 340, and tubing 321, 322, 323. The syringe 320 is connected with a first section of tubing 321 (e.g., medical fluid tubing) to a delivery side of the nasal insert 310 to deliver saline 30 to the patient’s nasal cavity 302, and a second section of tubing 322 connects a return side of the nasal insert 310 to the collection container 340 to deliver the sample 31 to the collection container 340 (indicated by arrow 395). The third section of tuning 323 is connected to the collection container 340 and positioned to be reached by the patient’s mouth 304 in order for the patient to apply suction (indicated by arrow 305) to the collection container 340.

FIG. 3A shows the nasal insert 310 disposed in the nasal opening 303 of the patient’s head 301. In operation, and after insertion of the nasal insert 301 into the nasal opening 303, the patient (or another operator) uses a plunger 325 of the syringe 320 to inject saline 30 from the syringe (indicated by arrow 391) though the first section of tubing 321, into the nasal insert 310, where it is sprayed (indicated by arrows 392) towards and against the olfactory region (as shown in FIG. 1) of the patient’s nasal cavity 302. Sample saline 31 (i.e., saline 30 after contacting the olfactory region) returns to a section port (shown more clearly in FIG. 3B) of the nasal insert 310 where it is collected and delivered to the collection container 340 via the second section of tubing 322. The sampled saline 31 is, in some instances, collected in the collection container 340 using one or more of the following methods: First, gravity aided return, which is aided by the presence of a level 341 in the collection container 340 to assist the patient in keeping the angle of the head pointed downwards towards the collection container 340. Second, the application of oral suction that requires closure of the soft palate. As illustrated in FIG. 3 A, the patient’s soft palate 306 must close (as indicated by arrow 394) to enable the patient to apply suction. The patient seals the lips of their mouth 304 around the third section of tubing 323 and applies suction to the collection container 340 (and therefore to the suction port of the nasal insert 310). This not only prevents loss of the sampled saline down the nasopharynx but improves the recovery of the sampled saline into the collection container 340. In some instances, the collection container 340 includes a valve 342 to prevent backflow of the lavage into the mouth once the collection container 340 is filled. In some instances, the collection container 340 contains a preservative prior to use. In some instances, the collection container 340 contains a protease inhibitor or a nuclease inhibitor, e.g., an RNAse inhibitor (e.g., RNAlater). After collection of the sample saline 31 in the collection container 340, a cap 343 of the collection container 340 is removed and a separate sealing cap (not illustrated) is attached to the collection container 340 before returning the collection container 340. In some instances, the collection container 340, once sealed after use, is configured to be mailed to a location with an ice pack to further preserve the sampled saline 31 prior to processing.

FIG. 3B is an illustration of the nasal insert 310 of the sampling device 300 of FIG. 3A. FIG. 3B shows the nasal insert 310 having the first and second sections of tubing 321,322 connected to the underside of a conical insertion portion 313 of the nasal insert 310. The top of the conical insertion portion 313 defines an aperture 311 which is, in some instances, a tip aperture defining a spray nozzle and configured to direct a spray of saline 31 (indicated by arrows 392) towards the olfactory mucosa region of the patient’s nasal cavity 302. The conical insertion portion 313 also defines a suction port 312 arranged on the side of the conical insertion portion 313. The conical insertion portion 313 includes an outwardly extending rim region 314 that widens the nasal insert at the end proximal to the aperture 311. The rim 314 and is, in some instances, sized and shaped to control the entry depth of the conical insertion portion 313 into the patient’s nasal opening 303, and, in some instances, the shape of the conical insertion portion 313 together with the rim 314 are arranged to maintain the direction of the spray 392 from the aperture 311 and position the suction port 312 at a level of the nasal floor 361 of the nasal cavity (302 of FIG. 3 A) of the patient when the nasal insert 310 is placed into the nasal opening 303. In operation, the flow 391 of saline from the first section of tubing 321 passes into the nasal insert 310 (as indicated by arrow 391) as is expelled from the aperture 311, as discussed above. The suction port 312 is in fluid communication with the second section of tubing 322 and is delivers a negative pressure to the nasal cavity 302 when the patient applies suction to the third section of tubing (323 of Fig. 3A). In some instances, gravity alone delivers a flow of sample saline 31 to the suction port 312 where it is delivered (arrow 394) to the collection container 340.

Kits

Also provided herein are kits for use in the methods described herein. For example, the kits can include one or more of: a sponge for sample collection; a delivery device for placing the sponge; a collection container; a protease inhibitor and/or RNAse inhibitor (e.g., RNAlater) for sample preservation, optionally provided within the collection container; sterile saline; and a cold or freezer pack for specimen preservation during shipment.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Materials and Methods for CDOME collection, isolation, and protein yield

The following materials and methods were used for CDOME collection, isolation, and protein yield detection.

CDOME Collection : A low volume concentrated collection strategy was used for CDOME collection. The low volume strategy included the placement of a compressed sterile 2x2x5mm poly -vinyl-alcohol sponge within the olfactory cleft. As the sponge expanded, it absorbed the olfactory mucus which was then extracted through centrifugation as described below. The sponge was stored at -80°C in protease inhibitor until isolation.

Exosome Purification from Whole Mucus: The exosome purification procedure was adapted from the ultracentrifugation (UCF) procedure described by Thery et al[l 8] . This technique was compared with a commercially available precipitation method (ExoQuick™, System Biosciences, Palo Alto, CA) and provided greater purity with higher protein and exosome yield in agreement with van Deun et al. [19] Mucus samples were extracted from the PVA sponge by centrifugation (1500 g at 4^°C for 30 minutes). The mucus was then diluted in 150 pL of lx phosphate buffered saline (PBS, Life Technologies, Carlsbad, CA) with Protease Inhibitor Cocktail (1: 100, Sigma, St. Louis, MO). Cellular debris was pelleted by centrifugation at 45 min at 12,000 x g at 4^°C. The supernatant was then suspended in 4.5mL of PBS in polypropylene tubes (Thinwall, 5.0 mL, 13 x 51 mm, Beckman Coulter,

Indianapolis, IN) and ultracentrifuged for 2 hours at 110,000 x g, at 4^°C. The supernatant was collected and the pellet was resuspended in 4.5 mL lx PBS. The suspension was filtered through a 0.22-pm filter (Fisher Scientific, Pittsburgh, PA) and collected in a fresh ultracentrifuge tube. The filtered suspension was then centrifuged for 70 min at 110,000 x g at 4^°C. The supernatant was collected and the pellet was resuspended in 200 pl PBS with protease inhibitor. Prior to cell culture dosing the exosome concentration of each pellet was determined using a

commercially available enzyme linked immunosorbent assay (ELISA) for the established exosome markers CD63 and CD9 (ExoELISA, System Biosciences, Palo Alto, CA) as previously described [20] The ratio of exosomes to total protein, as well as the presence of contaminating endoplasmic reticulum derived proteins Calnexin and Argonaut 2, was used to determine CDOME yield and purity.

Transmission Electron Microscopy of Mucus Derived Exosomes: The exosome transmission electron microscopy (TEM) procedure was adapted from Thery et al. [18] Isolated exosomes were fixed for 1 hour at room temperature in 2% paraformaldehyde in 0.1M sodium phosphate buffer (Electron Microscopy Sciences, Hatfield, PA). 5 pL of the exosomes were absorbed on to Formvar-carbon coated electron microscopy grids (Electron Microscopy Sciences) for 20 minutes. After absorption, the grids were rinsed in PBS 3 times and then transferred to PBS/50 mM glycine (Sigma Aldrich, St. Louis MO) for 4 washes. The grids were blocked in 5% Bovine Serum Albumin (BSA, Fisher Scientific) in lx phosphate buffered saline (buffer) for 10 minutes at room temperature. The grids were incubated at 4°C overnight in the primary antibody (1:25, Purified Mouse Anti-Human CD63 Clone H5C6, BD Biosciences) diluted in 1% BSA buffer. The grids were then rinsed in

0.1% BSA buffer and then 0.5% BSA buffer 6 times each. Then the secondary Protein-G antibody (1:20 in l%BSA buffer, EM Grade, lOnm, Electron Microscopy Services, Hatfield, PA) in 5% BSA buffer was applied for 1 hour at room temperature and rinsed 8 times with lx PBS. The grids were incubated in 1% glutaraldehyde in 0.1M sodium phosphate buffer (Electron Microscopy Services) for 5 minutes. After rinsing 8 times in deionized water, the grids were contrasted in uranyl-oxalate solution, pH 7 (UA, Electron Microscopy Services) for 5 minutes. The grids were blotted on filter paper and air dried prior to imaging. The exosomes were observed using a FEI Tecnai G2 Spirit transmission electron microscope (FEI, Hillsboro, Oregon) at an accelerating voltage of 100 kV interfaced with an AMT XR41 digital CCD camera (Advanced Microscopy Techniques, Woburn, Massachusetts) for digital TIFF file image acquisition. Rabbit IgG (Vector Laboratories, Burlingame, CA) and CD63 lysate (Novus Biologicals CD63 Overexpression Lysate (Native), Fisher Scientific) were used as negative and positive controls, respectively.

In vivo Quantification of Mucus Derived Exosomal Protein Concentration :

Mucus was collected from both control and CRSwNP patients for in vivo

characterization of exosomal concentrations of beta-2 transferrin, phospho-tau (pS396), total tau, and Abeta 1-42. The mucus was collected using a PVA sponge followed by exosome purification as described above. The purified exosome fraction was subjected to beta-2 transferrin, phospho-tau (pS396), total tau, Abeta 1-42, CD63, and CD9 (Systems Bioscience) ELIS As to determine their relative concentrations within the purified exosomal fraction. All values were normalized to the total protein concentration within the same sample using a Micro BCA Protein Assay Kit (Pierce, Rockford, IL).

Statistical Analysis of CDOME derived biomarkers: The statistical significance of differences between group means for patients with AD and controls was determined using an unpaired t test including a Bonferroni correction in the interpretation. Separate discriminant classifier analyses is conducted to define the best simple linear models for comparing AD biomarkers with controls. Receiver operating characteristics (ROC) analyses are conducted under a parametric or nonparametric distribution assumption for standard error of area to determine the performance of models for discriminating AD and controls. Discriminant and ROC analyses are conducted with SAS (SAS Institute Inc., Cary, NC).

Example 1. CSF-derived Biomarkers of AD are present in CDOMEs

CDOMEs were collected from normal individuals and characterized using TEM (see Figure 2A, which shows whole mounted exosomes purified from nasal mucus, and Figure 2B, a Negative control (bar lOOnm for b-d)), confirming the typical exosome size and morphology.

CDOME-derived Ab1-42, P S396 x, P-T181-X, total x, total protein, CD63, and CD9 were quantified using commercially available ELISA (Thermo Scientific and System Biosciences). The results are shown in Figures 2C-D. Figure 2C demonstrated the presence of B2-transferrin, a protein only found in CSF, in

CDOMEs. Direct sampling of the olfactory mucus produced greater exosomal protein yield than sampling mucus from within the nasopharynx, which has been diluted by sinonasal exosomes (see Figure 1).

Figure 2D demonstrates the presence of p-tau S396, total tau, and AB 1-42 in CDOMEs. Again, biomarkers from CDOMEs directly derived from the olfactory mucosa were more concentrated than those sampled from the nasopharynx.

Example 2: Exploratory proteomic analysis to identify novel potential AD biomarkers

CDOME Proteomic Array. CDOMEs are collected and isolated by group as described above. Samples are subjected to proteomic analysis using the Slow Off-rate Modified Aptamer (SOMAmer)-based capture array called‘SOMAscan’ (SomaLogic, Inc, Boulder, Colorado). Quality control is performed at the sample and SOMAmer level using control SOMAmers on the microarray and calibration samples. At the sample level, hybridization controls on the microarray will be used to monitor sample-by-sample variability in hybridization, while the median signal over all SOMAmers will be used to monitor overall technical variability. The resulting hybridization scale factor and median scale factor will be used to normalize data across samples. The acceptance criteria for these values are 0.4-2.5, based on historical trends in these values [27] Somamer by somamer calibration occurs through the repeated measurement of calibration samples, these samples are of the same matrix as the study samples, and are used to monitor repeatability and batch to batch variability. Historical values for these calibrator samples for each SOMAmer are used to generate a calibration scale factor. The acceptance criteria for calibrator scale factors is that 95% of SOMAmers must have a calibration scale factor within ±0.4 of the median.

SOMAscan Analysis: Protein levels are log transformed and tested to verify normality. Protein values between groups are compared using ANOVA with a p-value of 0.05 set as the significance threshold, without adjustment for multiple comparisons. False discovery rate (FDR) multiple testing corrections are applied to the resulting p- values; with both a strict (FDR q-value of 0.05) and less strict significance level used (FDR q-value of 0.1). This will enable both strong and promising results to be detected respectively. REFERENCES

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OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting a neurological disease in a subject, the method comprising: isolating CSF Derived Olfactory Mucus Exosomes (CDOMEs) from the subject; determining a level of a biomarker associated with a neurological disease in the CDOMEs; and

diagnosing a neurological disease in the subject based on the level of the biomarker associated with the neurological disease in the CDOMEs from the subject.

2. A method of diagnosing Alzheimer’s Disease (AD) in a subject, the method

comprising:

isolating CSF Derived Olfactory Mucus Exosomes (CDOMEs) from the subject; determining a level of a biomarker associated with AD in the CDOMEs; and diagnosing AD in the subject based on the level of the biomarker associated with the neurological disease in the CDOMEs from the subject.

3. The method of claim 1 or 2, further comprising comparing the level of the biomarker in the CDOMEs to a reference level, and diagnosing the disease when the level of the biomarker in the CDOMEs is above the reference level.

4. The method of claim 2 or 3, wherein the marker is selected from the group consisting of Ab 1-42, Ab 1-42/Ab 1-40 ratio, P-S396-T, R-T181-t, and total t.

5. The method of claim 1, further comprising selecting and optionally administering a treatment for the neurological disease to the subject.

6. The method of claim 2, further comprising selecting and optionally administering a treatment for AD to the subject.

7. A method of monitoring the efficacy of a treatment for a neurological disease,

preferably Alzheimer’s Disease (AD), in a subject, the method comprising:

isolating a first sample comprising CSF Derived Olfactory Mucus Exosomes

(CDOMEs) from the subject;

determining a level of a biomarker associated with the neurological disease in the first sample;

treating the subject;

isolating a second sample comprising CSF Derived Olfactory Mucus Exosomes (CDOMEs) from the subject;

determining a level of the biomarker associated with neurological disease in the second sample;

and

identifying the treatment as successful in a subject when the level of the biomarker in the second sample is below the level of the biomarker in the first sample.

8. A nasal cavity sampling device comprising:

a nasal insert sized and shaped to be inserted into a nasal opening of a nasal cavity of a patient, the nasal insert being configured to receive a first flow of medical fluid from a syringe, the nasal insert comprising:

a first aperture arranged at a distal end of the nasal insert and configured to deliver a medical fluid along a trajectory towards an olfactory region in the nasal cavity of the patient, and

a second aperture arranged proximal to the first aperture and configured to receive a second flow of medical fluid from the nasal cavity of the patient; and

a collection container fluid coupled to the nasal insert and configured to receive the second flow of the medical fluid from the second aperture of the nasal insert.

9. The nasal cavity sampling device of claim 8, comprising: a syringe fluidly coupled to the nasal insert and configured to deliver the first flow of the medical fluid to the first aperture of the nasal insert, the syringe containing the medical fluid.

10. The nasal cavity sampling device of claim 8, where the medical fluid is saline.

11. The nasal cavity sampling device of claim 8, where the collection container contains a preservative.

12. The nasal cavity sampling device of claim 8, where the collection container comprises a tube extending from the collection container and arranged to be disposed in the patient’s mouth in order for the patient to apply suction to the collection container, the suction drawing the medical fluid from the second aperture and into the collection container.

13. The nasal cavity sampling device of claim 9, where the tube comprises a valve

configured to maintain a negative pressure in the collection container after the patient’s application of suction.

14. The nasal cavity sampling device of claim 8, where the collection container

comprises a level configured to aid the patient in positioning the collection container at a given level with respect to the nasal insert.

15. The nasal cavity sampling device of claim 8, where the nasal insert comprises a

conical surface sized and positioned to expose the first aperture to the olfactory region when the nasal insert is disposed in the nasal opening, the conical surface including the first and second apertures.

16. The nasal cavity sampling device of claim 12, where conical surface is sized and positioned to dispose the second aperture along a nasal floor of the nasal cavity when the nasal insert is disposed in the nasal opening.

17. The nasal cavity sampling device of claim 12, where the conical surface is sized and positioned to sealingly interface with the nasal opening of the patient when the nasal insert is disposed in the nasal opening.

18. A kit for use in a method described herein, comprising one or more of: a sponge for sample collection; a delivery device for placing the sponge; a collection container; a protease inhibitor and/or RNAse inhibitor for sample preservation; sterile saline; and a cold or freezer pack for specimen preservation during shipment.