Protein markers for assessing and treating neurodegenerative diseases

Abstract

The present invention provides a protein marker, Nell-1, present in an individual's blood sample in an amount that is associated with neurodegenerative disorders such as Alzheimer's disease (AD), mild cognitive impairment (MCI), and Parkinson's disease (PD). Corresponding diagnostic or therapeutic methods for diagnosing or treating these neurodegenerative disorders, as well as kits for diagnosing or treating said neurodegenerative disorders, are also provided.

Description

[Technical Field]

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 413,969, filed October 7, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0003] Brain diseases, such as neurodegenerative and neuroinflammatory disorders, are devastating illnesses that affect a significant portion of the population. Many are difficult to cure, severely debilitating, and often result in progressive deterioration of brain structure and function over time. Because older adults are at higher risk of developing these diseases, the prevalence of these diseases is rapidly increasing as the global elderly population grows. Currently, many neurodegenerative and neuroinflammatory diseases are difficult to diagnose due to limited understanding of their pathophysiology. Meanwhile, current treatments are ineffective and do not meet market demand, which is increasing significantly each year due to the aging population. For example, Alzheimer's disease (AD) is characterized by a gradual but steady decline in learning and memory function and is one of the leading causes of death among older adults. The increasing prevalence of AD has fueled the need and demand for better diagnostics. According to Alzheimer's Disease International, approximately 40 million people currently suffer from AD, a number predicted to triple over the next 30 years. China has one of the fastest-growing elderly populations. Population projections indicate that by 2030, one in four people will be 60 years of age or older, and the majority of these people will be at risk of developing Alzheimer's disease. Indeed, the number of AD cases in China doubled from 3.7 million to 9.2 million between 1990 and 2010, and is projected to reach 22.5 million by 2050. Hong Kong's population is also aging rapidly. Those aged 65 years or older are projected to account for 24% of the population by 2025 and 39.3% by 2050. The number of AD cases is expected to exceed 330,000 by 2039.

[0004] Even more concerning is the fact that despite the increasing prevalence of AD, many people do not receive an accurate diagnosis. According to the Alzheimer's Disease International World Alzheimer's Report 2015, even in high-income countries, only 20–50% of dementia cases are diagnosed in primary care. The remainder remain undiagnosed or misdiagnosed. This "treatment gap" is even more severe in low- and middle-income countries. Without a formal diagnosis, patients cannot receive the treatment and care they need, and patients or their caregivers are ineligible for important support programs. Early diagnosis and early intervention are two important means of narrowing the treatment gap. Therefore, early diagnostic tools that can rapidly and accurately determine disease risk have significant therapeutic value in various aspects. Research has confirmed that AD affects the brain well before actual symptoms of memory loss or cognitive decline actually appear. However, to date, few diagnostic tools exist for early detection (see, for example, International Publication No. WO 2021 / 228125). In most cases, by the time a patient is diagnosed with AD by currently commonly used diagnostic methods, including subjective clinical assessment, the pathological symptoms are usually already at an advanced stage. Therefore, there is an urgent need to develop new and more effective methods for early diagnosis of AD in patients or for detecting an increased risk of developing AD in the future, with the aim of improving AD treatment and long-term management. The present invention addresses this and other related needs by disclosing novel methods and kits related to using the circulating protein Nell-1 as a biomarker for individualized assessment of the risk of developing neurodegenerative disorders such as Alzheimer's disease (AD), mild cognitive impairment (MCI), and Parkinson's disease (PD), and as a therapeutic target for treating neurodegenerative disorders.

[0005] Summary of the Invention [Means for solving the problem]

[0006] The present invention relates to the discovery of novel plasma protein markers associated with neurodegenerative disorders such as Alzheimer's disease (AD), mild cognitive impairment (MCI), and Parkinson's disease (PD). Accordingly, the present invention provides methods and compositions useful for diagnosing these neurodegenerative disorders and determining the therapeutic efficacy of therapeutic agents for neurodegenerative disorders. Accordingly, in a first aspect, the present invention provides a method for assessing a subject's risk of developing a neurodegenerative disorder such as AD, MCI, or PD in the future. The method comprises the following steps: First, comparing the level of Nell-1 protein in the subject's plasma, serum, or whole blood with a standard control level of Nell-1 protein found in the plasma, serum, or whole blood of an average healthy subject who is not afflicted with or at increased risk of a neurodegenerative disorder; Second, detecting a decrease in the level of Nell-1 protein in the subject's plasma, serum, or whole blood from the standard control level, thereby determining the subject as afflicted with or at increased risk of the neurodegenerative disorder. Alternatively, as an alternative to the second step, a third step is to detect that the level of Nell-1 protein in the subject's plasma, serum, or whole blood is not decreased from the standard control level, thereby determining that the subject is not suffering from or at increased risk of the neurodegenerative disorder. In some embodiments, the method further comprises a step of measuring the level of Nell-1 protein in the plasma, serum, or whole blood before the first step. In some embodiments, the method further comprises a step of obtaining a plasma, serum, or whole blood sample from the subject before the measuring step.In some embodiments, the method further comprises, after the third step, comparing the level of Nell-1 protein in the plasma, serum, or whole blood of the subject measured at a later time point (i.e., a second time point) with the level of Nell-1 protein in the plasma, serum, or whole blood of the subject at the first step (i.e., the original time point, or an earlier or first time point), wherein a higher level of Nell-1 protein in the plasma, serum, or whole blood measured at the later time point indicates an improvement in the neurodegenerative disorder, and a lower level of Nell-1 protein in the plasma, serum, or whole blood measured at the later time point indicates a worsening of the neurodegenerative disorder. In some embodiments, between the third step (or the first time point) and the later time point (or the second time point), the subject has been administered a therapeutic agent intended to treat the neurodegenerative disorder. In some embodiments, the neurodegenerative disorder for which risk is being assessed is Alzheimer's disease (AD). In some embodiments, the neurodegenerative disorder being evaluated is mild cognitive impairment (MCI) or Parkinson's disease (PD).

[0007] In a second aspect, the present invention provides a method for assessing the relative risk level for a neurodegenerative disorder, such as AD, MCI, or PD, between two or more subjects. The method includes the following steps: first, comparing the level of Nell-1 protein in the plasma, serum, or whole blood of a first subject with the level of Nell-1 protein in the plasma, serum, or whole blood of a second subject; second, detecting a lower level of Nell-1 protein in the plasma, serum, or whole blood of the second subject than the level of Nell-1 protein in the plasma, serum, or whole blood of the first subject; and third, determining that the second subject has a lower severity or lower risk of the neurodegenerative disorder than the first subject. In some embodiments, the neurodegenerative disorder for which risk is assessed is Alzheimer's disease (AD). In some embodiments, the neurodegenerative disorder for which risk is assessed is mild cognitive impairment (MCI) or Parkinson's disease (PD). In some embodiments, the claimed method further comprises measuring the level of Nell-1 protein in plasma, serum, or whole blood prior to the first step. In some embodiments, the above-described method further comprises obtaining a sample of plasma, serum, or whole blood from the subject prior to the measuring step.

[0008] In a third aspect, the present invention provides kits for assessing the risk of neurodegenerative disorders, such as AD, MCI, and PD, in a subject, or for assessing the therapeutic efficacy of a treatment regimen for these neurodegenerative disorders. The kits include a first container containing a first reagent capable of determining the level of Nell-1 protein in the subject's plasma, serum, or whole blood, and optionally a second container containing a second reagent capable of determining the level of phosphorylated tau-181 (pTau181) or neurofilament light polypeptide (NfL) in the subject's plasma, serum, or whole blood. In some embodiments, the kits include reagents capable of determining the levels of two proteins, pTau181 and NfL, in the subject's plasma, serum, or whole blood. In some embodiments, the kits further include a standard control reflecting the level of Nell-1 protein found in the plasma, serum, or whole blood of an average healthy subject not afflicted with and not at increased risk of a neurodegenerative disorder. In some embodiments, the kits described above or herein are intended for assessing the risk and therapeutic efficacy of treatment regimens for the neurodegenerative disorders Alzheimer's disease (AD), mild cognitive impairment (MCI), or Parkinson's disease (PD).

[0009] In a fourth aspect, the present invention provides a method for treating a neurodegenerative disorder or reducing the risk of such a disorder. The method comprises administering to a subject in need thereof (1) an effective amount of a Nell-1 enhancer that enhances the expression or activity of a Nell-1 protein, or (2) an effective amount of a Nell-1 receptor enhancer that enhances the expression or activity of a Nell-1 receptor. In some embodiments, the Nell-1 enhancer is a Nell-1 protein or a nucleic acid encoding a Nell-1 protein. In some embodiments, the Nell-1 enhancer is a Nell-1 receptor, a nucleic acid encoding a Nell-1 receptor, or an agonist of a Nell-1 receptor. In some embodiments, the administering step comprises brain-targeted delivery of the Nell-1 enhancer or Nell-1 receptor enhancer. In some embodiments, when a Nell-1 protein or a nucleic acid encoding a Nell-1 receptor is administered, the nucleic acid is formulated in a lipid nanoparticle composition for delivery. In some embodiments, the nucleic acid encoding a Nell-1 protein or a Nell-1 receptor is introduced by gene editing. In some embodiments, the claimed method of treatment further comprises measuring the level of Nell-1 protein in the subject's plasma, serum, or whole blood before and / or after the administering step. In some embodiments, the claimed method further comprises obtaining a plasma, serum, or whole blood sample from the subject prior to the one or more measuring steps. In some embodiments of this method of treatment, the neurodegenerative disorder being treated is Alzheimer's disease (AD), mild cognitive impairment (MCI), or Parkinson's disease (PD).

[0010] In a fifth aspect, the present invention provides a method for evaluating the effectiveness of a therapeutic agent for treating a neurodegenerative disorder in a subject. The method comprises the steps of: (1) comparing the level of Nell-1 protein in the plasma, serum, or whole blood of the subject before and after administering the therapeutic agent to the subject; (2) detecting an increase in the level of Nell-1 protein in the plasma, serum, or whole blood of the subject after administration of the therapeutic agent; and (3) determining that the therapeutic agent is effective in treating the neurodegenerative disorder. In some embodiments, the neurodegenerative disorder being treated is Alzheimer's disease (AD), mild cognitive impairment (MCI), or Parkinson's disease (PD). In some embodiments, the claimed method further comprises, prior to step (1), measuring the level of Nell-1 protein in the plasma, serum, or whole blood before and after administration. In some embodiments, the method further comprises, prior to the measuring step, obtaining plasma, serum, or whole blood samples from the subject before and after administration.

[0011] In some embodiments of the methods described above and herein, one or more subjects being tested for or treated for a neurodegenerative disorder (e.g., AD, MCI, or PD) or risk thereof are of Chinese descent. [Brief explanation of the drawings]

[0012] [Figure 1] Decreased levels of Nell-1 protein in blood distinguish AD patients from cognitively normal subjects. Figure 1a: Boxplot showing plasma Nell-1 levels in cognitively normal (CN) subjects and individual patients with Alzheimer's disease (AD) in the Hong Kong Chinese cohort (n=119CN+152AD). Statistical analysis was performed by linear regression adjusting for age, sex, and history of cardiovascular disease. β=-0.158, *P<0.05. Figure 1b: Receiver operating characteristic (ROC) curve and corresponding area under the curve (AUC) showing the classification performance of AD phenotypes by plasma Nell-1 levels in the Hong Kong Chinese cohort.

[0013] [Figure 2] Decreased levels of Nell-1 protein in blood distinguish MCI patients from cognitively normal subjects. Figure 2a: Boxplot showing plasma Nell-1 levels in cognitively normal (CN) subjects and individual patients with mild cognitive impairment (MCI) in the Hong Kong Chinese cohort (n = 119 CN + 109 MCI). Statistical analysis was performed by linear regression adjusting for age, sex, and history of cardiovascular disease. β = -0.187, *P < 0.05. Figure 2b: Receiver operating characteristic (ROC) curve and corresponding area under the curve (AUC) showing the classification performance of MCI phenotypes by plasma Nell-1 levels in the Hong Kong Chinese cohort.

[0014] [Figure 3] Decreased levels of Nell-1 protein in the blood distinguish PD patients from cognitively normal subjects. Figure 3a: Boxplot showing Nell-1 levels in the plasma of cognitively normal (CN) subjects and individual patients with Parkinson's disease (PD) in the Hong Kong Chinese cohort (n=119CN+39PD). Statistical analysis was performed by linear regression adjusted for age and sex. β=-0.370, ***P<0.001. Figure 3b: Receiver operating characteristic (ROC) curve and corresponding area under the curve (AUC) showing the classification performance of PD phenotypes by plasma Nell-1 levels in the Hong Kong Chinese cohort.

[0015] [Figure 4]Decreased blood levels of Nell-1 protein predict neurodegeneration, AD progression, cognitive decline, and aging. Figure 4a: Scatter plot and regression line (red) showing the association between plasma Nell-1 levels and neurodegeneration, as indicated by plasma NfL levels, in a Hong Kong Chinese cohort (n=315). Statistical analysis was performed by linear regression adjusting for age, sex, and disease diagnosis. β=-0.005. Figure 4b: Scatter plot and regression line (red) showing the association between plasma Nell-1 levels and Alzheimer's disease (AD) progression, as indicated by plasma pTau181 levels, in a Hong Kong Chinese cohort (n=300). Statistical analysis was performed by linear regression adjusting for age, sex, and disease diagnosis. β=-0.043. Figure 4c: Scatter plot and regression line (red) showing the association between cognitive function as measured by MoCA score and plasma Nell-1 levels in the Hong Kong Chinese cohort (n=384). Statistical analysis was performed by linear regression adjusting for gender, years of education, and disease diagnosis. β=0.012. Figure 4d: Scatter plot and regression line (red) showing the association between plasma Nell-1 levels and age in the Hong Kong Chinese cohort (n=424). Statistical analysis was performed by linear regression adjusting for gender and disease diagnosis. β=-0.014. R2 is Pearson's correlation coefficient.

[0016] [Figure 5]Mendelian randomization analysis identifies decreased plasma Nell-1 protein levels as a contributing factor to neurodegeneration. Figure 5a: Manhattan plot showing genetic variants associated with plasma Nell-1 levels in a Hong Kong Chinese cohort. The x-axis indicates the location of the genetic variant on chromosome 11, and the y-axis indicates the significance of the association. The red dashed line indicates the significance threshold (P = 0.01). Statistical analysis was performed by linear regression adjusting for age, sex, disease diagnosis, and demographic structure. cM / Mb, centimorgans per megabase. Figure 5b: Scatter plot and regression line (blue) showing the causal relationship between plasma Nell-1 levels and plasma NfL levels in a Hong Kong Chinese cohort (n = 255). Statistical analysis was performed by two-stage least squares regression adjusting for age, sex, disease diagnosis, and demographic structure.

[0017] [Figure 6] Treatment with Nell-1 protein promotes synapse formation in hippocampal neurons. Immunohistochemical analysis of excitatory synapse density in primary cultured rat hippocampal neurons treated with Nell-1 (2 μg / mL, 48 hours) or DPBS (control). Figure 6a: Immunostaining for VGluT1 (red, presynaptic marker), PSD-95 (green, postsynaptic marker), and MAP2 (blue, dendritic marker). Scale bar: 20 μm. Figure 6b: High-magnification image of a dendrite (within the white rectangle in Figure a) showing excitatory synapses (VGluT1-positive PSD-95 clusters, white arrowheads). Scale bar: 5 μm. Figure 6c: Bar graph showing quantification of VGluT1-positive PSD-95 clusters. (Statistical analysis was performed by two-tailed unpaired t-test (***P<0.001). Control group: n = 15 neurons, Nell-1 group: n = 14 neurons, from two independent experiments.)

[0018] [Figure 7]Treatment with Nell-1 protein reverses impaired hippocampal synaptic plasticity in APP / PS1 AD transgenic mouse models. Electrophysiological analysis of long-term potentiation (LTP) in 11-month-old wild-type (WT) and APP / PS1 mice treated with Nell-1 (5 ng / day, intracerebroventricular delivery, 7 days) or artificial cerebrospinal fluid (ASF). LTP in the hippocampal CA1 region is induced by three high-frequency stimulations (HFS). Figure 7a: Mean slope of baseline-normalized excitatory field postsynaptic potentials (fEPSPs). Figure 7b: Bar graph showing normalized fEPSP slopes 60 min after HFS stimulation. (Statistical analysis was performed by one-way ANOVA and Tukey's post-hoc test (*P<0.05). WT control group: 10 brain sections obtained from n = 4 mice, WT Nell-1 group: 14 brain sections obtained from n = 6 mice, APP / PS1 control group: 10 brain sections obtained from n = 5 mice, APP / PS1 Nell-1 group: 11 brain sections obtained from n = 4 mice, from two independent experiments.) Data are shown as mean ± SEM.

[0019] [Figure 8] Treatment with Nell-1 protein increases hippocampal neurogenesis in mice. Immunohistochemical analysis of immature neurons in the dentate gyrus (DG) region of the hippocampus in 11-month-old wild-type (WT) mice treated with Nell-1 (5 ng / day, intracerebroventricular delivery, 7 days) or artificial cerebrospinal fluid (ASF). Figure 8a: Immunostaining for DCX (cyan, immature neuron marker). Scale bar: 100 μm. Figure 8b: High-magnification image of the DG region (within the white rectangle in Figure a) showing immature neurons (white arrowheads). Scale bar: 50 μm. Figure 8c: Bar graph showing quantification of immature neurons in the subgranular zone of the DG. (Statistical analysis was performed by unpaired t-test between two groups (*P<0.05). Control group: n = 3 mice, Nell-1: n = 3 mice.) DETAILED DESCRIPTION OF THE INVENTION

[0020] definition

[0021] As used herein, the term "Nell-1" refers to the protein kinase C-binding protein NELL1, which is also known as Nel-associated protein 1 or neuroepithelial growth factor-like 1 protein. One exemplary Nell-1 protein has UniProtKB accession number Q92832, however, the term "Nell-1" also encompasses all variants to this exemplary sequence.

[0022] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single- or double-stranded form, and polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J.Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used to encompass the subject matter defined by the terms gene, cDNA, and mRNA encoded by a gene.

[0023] The term "gene" refers to a segment of DNA involved in producing a polypeptide chain. A gene may include intervening sequences (introns) between individual coding segments (exons), in addition to regions preceding and following the coding region (leader and trailer).

[0024] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. All three terms apply to one or more amino acid residues, amino acid polymers that are artificial chemical mimetics of corresponding naturally occurring amino acids, and natural and non-natural amino acid polymers. As used herein, these terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0025] In this disclosure, the term "biological sample" or "sample" includes sections of tissue such as biopsy and autopsy samples, frozen sections taken for histological purposes, or processed forms of any of these samples. Biological samples include blood and blood fractions or products (e.g., whole blood, acellular fractions of blood (serum, plasma), and blood cells), sputum or saliva, lymphoid and tongue tissue, cultured cells (e.g., primary cultures, explants, and transformed cells), feces, urine, stomach biopsy tissue, and the like. Biological samples are typically obtained from eukaryotic organisms, which may be mammalian, primate, or human subjects.

[0026] The terms "immunoglobulin" or "antibody" (used interchangeably herein) refer to an antigen-binding protein that has the ability to specifically bind to an antigen and has a basic structure consisting of four polypeptide chains: two heavy chains and two light chains. The chains are stabilized, for example, by interchain disulfide bonds. Both the heavy and light chains are folded into domains.

[0027] The term "antibody" also refers to antibody fragments (e.g., Fab fragments) that bind to antigens and epitopes, and can be used in immunoaffinity assays. Several well-characterized antibody fragments exist. For example, pepsin digests the C-terminal side of the disulfide bond in the hinge region of an antibody to produce F(ab)'2. F(ab)'2 is a dimer of Fab, which itself is a light chain and a V linked by a disulfide bond. H -C H1. F(ab)'2 can be reduced under mild conditions to cleave the disulfide bond in the hinge region and convert the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (for a more detailed description of other antibody fragments, see, for example, Fundamental Immunology, Paul, ed., Raven Press, NY (1993)). While various antibody fragments are defined by the digestion of whole antibodies, those skilled in the art will appreciate that these fragments can be synthesized de novo by chemical means or recombinant DNA technology. Thus, the term antibody also includes antibody fragments produced by the modification of whole antibodies or those synthesized using recombinant DNA technology.

[0028] The phrase "specifically binds," when used in the context of describing the binding relationship between a particular molecule and a protein or peptide, refers to a binding reaction that is determinative of the presence of that protein in a heterogeneous population of proteins and other biological materials. Thus, under defined binding assay conditions, a specific binding agent (e.g., an antibody) binds to the specific protein of interest at least twice as strongly as background and does not bind in any substantial, significant amount to other proteins present in the sample. Specific binding of an antibody under such conditions may require an antibody selected for its specificity for a particular protein or proteins and its lack of binding to similar "sister" proteins. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein or its particular forms. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a particular protein (see Harlow & Lane, Antibodies, A Laboratory Manual (1988) for immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective binding reaction is at least twice the background signal or noise, and typically 10 to 100 times the background. On the other hand, when the term "specifically binds" is used in the context of a polynucleotide sequence forming a double-stranded complex with another polynucleotide sequence, this term refers to "polynucleotide hybridization," i.e., binding based on Watson-Crick base pairing in a "polynucleotide hybridization method."

[0029] As used herein, "increase" or "decrease" refers to a detectable positive or negative quantitative change compared to a comparison control, e.g., an established standard control (e.g., the average level / amount of a particular protein found in samples from healthy subjects who have not been diagnosed with and are not at elevated risk for a neurodegenerative disorder such as AD, MCI, or PD). An increase is typically a positive change of at least 10%, alternatively at least 20%, at least 50%, or at least 100% compared to the control value, and in some cases may be at least 2-fold, at least 5-fold, or even 10-fold. Similarly, a decrease refers to a negative change, typically at least 10%, alternatively at least 20%, at least 30%, or at least 50% compared to the control value, and in some cases may be at least 80% or 90% or more. Other terms indicating a quantitative change or difference from a comparison standard, such as "more," "less," "higher," and "lower," are also used herein in a similar manner. In contrast, the terms "substantially the same" or "substantially unchanged" mean that there is little or no quantitative change from the standard control value, typically within ±10%, ±5%, ±2%, or less of the standard control value.

[0030] A "label," "detectable label," or "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include: 32 Detectable labels include haptens and proteins that can be made detectable by incorporating radioactive moieties into the protein, or that can be used to detect antibodies that specifically react with the protein. Detectable labels are usually attached to a probe or molecule with specific binding properties (e.g., an antibody with known binding specificity for a polypeptide antigen), making the presence of the probe (and therefore its binding partner) readily detectable.

[0031] The term "amount," as used herein, refers to the quantity of a substance of interest (e.g., a polypeptide of interest). Such quantity can be expressed as an absolute value (i.e., the total amount of that substance in a sample) or as a relative value (i.e., the concentration of that substance in a sample).

[0032] As used herein, the term "subject" or "subject in need of treatment" includes an individual diagnosed with a disease or condition (e.g., a neurodegenerative disorder such as AD, MCI, or PD) or seeking medical treatment due to risk (e.g., family history). Subjects also include individuals currently receiving treatment who wish to adjust their treatment regimen. Subjects in need of treatment or individuals in need of treatment include individuals who exhibit symptoms of a neurodegenerative disorder, such as AD, or individuals at risk for developing the neurodegenerative disorder or its symptoms. For example, subjects in need of treatment include individuals with a genetic predisposition or family history for the neurodegenerative disorder, individuals who have previously experienced associated symptoms, individuals who have been exposed to a precipitating agent or event, and individuals suffering from chronic or acute symptoms of the disease. A "subject in need of treatment" may be of any age.

[0033] The terms "inhibitor," "activator," and "modulator" of a target protein refer to inhibitory, activating, or modulating molecules, respectively, identified by in vitro or in vivo assays for protein binding or signal transduction, and include, for example, ligands, agonists, antagonists, and their homologs and mimetics. The term "modulator" includes inhibitors and activators. An inhibitor is, for example, a substance that partially or completely blocks, reduces, prevents, delays activation, inactivates, desensitizes, or downregulates the activity of a target protein. In some cases, an inhibitor binds directly or indirectly to the protein, such as a neutralizing antibody. As used herein, an inhibitor is synonymous with an inactivator and antagonist. An activator or agonist is an agent that stimulates, increases, promotes, enhances activation, sensitizes, or upregulates the activity of a target protein. Modulators include ligands or binding partners of target proteins, including modified naturally occurring ligands, synthetically designed ligands, antibodies and antibody fragments, antagonists, agonists, small molecules including sugar-containing molecules, siRNA, RNA aptamers, and the like.

[0034] As used herein, the term "treatment" or "treating" refers to the act of eliminating, alleviating, ameliorating, reversing, preventing, and / or delaying the onset or recurrence of any symptoms of a given medical condition. In other words, "treating" encompasses both therapeutic and prophylactic intervention for the condition.

[0035] As used herein, the term "effective amount" refers to the amount of a substance administered to produce a therapeutic effect. Such effect includes preventing, correcting, or inhibiting the progression of symptoms of a disease / disorder and its associated complications to a detectable extent. The exact amount will vary depending on the purpose of treatment, but can be determined by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0036] A "pharmaceutically acceptable" or "pharmacologically acceptable" excipient means a substance that is not biologically harmful or has other undesirable properties. That is, a non-medicinal excipient is a substance that does not cause any undesirable biological effects when administered to an individual in conjunction with a biologically active substance. Also, a non-medicinal excipient does not interact in a deleterious manner with any of the components of the composition in which it is contained.

[0037] The term "non-medicinal ingredient" refers to essentially auxiliary substances that may be present in the final formulation of the compositions of the present invention. For example, the term "non-medicinal ingredients" includes solvents, binders, disintegrants, excipients (diluents), lubricants, glidants (flow agents), compression aids, colorants, sweeteners, preservatives, suspending / dispersing agents, film-forming / coating agents, flavors, and printing inks.

[0038] The term "consisting essentially of," in the context of describing a composition containing one or more active ingredients, means that the composition does not contain any ingredients with biological activity similar to or related to any of the active ingredients, or ingredients that may enhance or inhibit their activity. However, one or more inactive ingredients, such as physiologically or pharmacologically acceptable excipients, may be present in the composition. For example, a composition consisting essentially of an active agent effective to enhance Nell-1 protein expression or activity in a subject is a composition that does not contain other agents that may have a detectable positive or negative effect on the same target process (i.e., Nell-1 protein expression or activity) or that may measurably increase or decrease the onset or symptoms of a neurodegenerative disorder (e.g., AD, MCI, or PD).

[0039] The term "about" refers to a range of ±10% of a given value. For example, "about 10" means 90% to 110% of 10, or a range of 9 to 11.

[0040] As used herein, the term "standard control" refers to a sample containing a predetermined amount of an analyte (e.g., a given DNA, mRNA, or protein) and is used to indicate the amount or concentration of the analyte. The sample is taken from an average healthy subject who is not afflicted with, and not at risk for, a particular disease or condition (e.g., a neurodegenerative disorder such as Alzheimer's disease (AD), mild cognitive impairment (MCI), or Parkinson's disease (PD)). When used in the context of describing a value, the term may simply refer to the amount or concentration of the analyte present in the "standard control" sample.

[0041] The term "average," when used in the context of describing healthy subjects who do not have and are not at risk for developing a relevant disease or disorder (e.g., a neurodegenerative disorder such as Alzheimer's disease (AD), mild cognitive impairment (MCI), or Parkinson's disease (PD)), refers to a characteristic, e.g., the level of the relevant protein in a sample (e.g., serum, plasma, or whole blood), representative of a randomly selected group of healthy humans who do not have and are not at risk for developing the disease or disorder. This selected group should include a sufficient number of human subjects so that the average amount or concentration of the analyte of interest among these individuals reflects, with reasonable accuracy, the corresponding profile in the general population of healthy people. Optionally, the selected group of subjects can be chosen to be matched or comparable in similar background, e.g., age, sex, ethnicity, medical history, etc., as the individual being tested for indication or risk of the relevant disease or disorder.

[0042] As used herein, the term "inhibit" or "inhibition" refers to a detectable negative effect on the level of a targeted biological process or biomarker (e.g., a protein). Typically, inhibition manifests as at least a 10%, at least a 20%, at least a 30%, at least a 40%, or at least a 50% decrease in one or more parameters indicative of a biological process, its downstream effect, or the level of a biomarker, when compared to a control in the absence of such inhibition. The term "enhance" or "enhancement" is similarly defined, except that it indicates a positive effect. That is, a positive change is at least 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 80%, at least a 100%, at least a 200%, at least a 300%, or more, compared to a control. The terms "inhibitor" and "enhancement" are used to describe agents that exhibit the aforementioned inhibitory or enhancing effects, respectively. Also used in this disclosure are the terms "increase," "decrease," "more," and "less," which mean a positive change of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 300%, or more in one or more predetermined parameters, or a negative change of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, or more in one or more predetermined parameters.

[0043] As used herein, the term "Chinese" refers to people of Han Chinese descent who have resided in and for at least the past three, four, five, six, seven, or eight generations, or the past 100, 150, 200, 250, or 300 years, in mainland China and the historical territories of China, including Hong Kong.

[0044] Detailed Description of the Invention I. Introduction

[0045] Alzheimer's disease (AD) is the most common form of dementia worldwide, accounting for 60-70% of all dementia cases. AD is an irreversible degenerative brain disease and one of the leading causes of death in the elderly. The hallmark of this disease is the deposition of extracellular β-amyloid (Aβ) plaques and intracellular neurofibrillary tangles, which cause declines in memory, reasoning, judgment, and motor function, with symptoms progressing over time.

[0046] Currently, an estimated 55 million people worldwide suffer from dementia, 60–80% of whom suffer from AD. This number is predicted to rise significantly to 153 million by 2050 due to increasing life expectancy. There is no cure for AD, and the disease's pathophysiology remains poorly understood. Currently, only six drugs have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of AD. However, these drugs only alleviate AD symptoms and do not address the underlying pathology. These drugs cannot reverse the condition or prevent further deterioration, and are ineffective in treating severe cases. Therefore, early diagnosis and early intervention are crucial for the management of AD. Research has confirmed that AD affects the brain long before actual symptoms of memory loss or cognitive decline appear. However, currently, there are no effective and reliable diagnostic tools for early detection of AD. By the time AD is diagnosed using currently widely used standard methods (including subjective clinical assessment), the pathological symptoms are already in an advanced stage. The present disclosure provides a diagnostic method for diagnosing the presence of a neurodegenerative disorder such as AD, MCI, or PD, or for assessing the risk of developing such a neurodegenerative disorder in the future, using the protein marker Nell-1 (also known as protein kinase C-binding protein NELL1, Nel-related protein 1, or neuroepithelial growth factor-like 1 protein). This allows for early diagnosis of the disorder, enabling early therapeutic intervention aimed at preserving or restoring cognitive function. Nell-1 protein can be used as both a therapeutic target and a surrogate marker for indicating the disease state.

[0047] II. Quantification of Marker Proteins

[0048] A. Sample Acquisition

[0049] The first step in practicing the present invention is to collect a blood sample from a subject to assess their risk of developing a neurodegenerative disorder (e.g., AD, MCI, or PD) or to monitor the severity or progression of a neurodegenerative disorder. The same type of sample should be collected from both the control group (cognitively normal individuals who are not affected by and do not have an increased risk of a neurodegenerative disorder) and the test group (e.g., subjects being tested for possible or increased risk of a neurodegenerative disorder such as AD, MCI, or PD). For this purpose, standard procedures routinely used in hospitals or clinics are followed.

[0050] To detect the presence or amount of a marker protein of interest or to assess the risk of developing a neurodegenerative disorder, a blood sample may be collected from an individual patient, and the level of the relevant marker protein (e.g., Nell-1 protein) in plasma, serum, or whole blood may be measured and compared with a standard control. If the level of Nell-1 protein (e.g., normalized level) is observed to be decreased compared to the control level, the test subject is considered to have or be at high risk of developing a neurodegenerative disorder such as AD, MCI, or PD in the future. To monitor disease progression or evaluate the effectiveness of treatment in patients with neurodegenerative disorders, blood samples may be collected from an individual patient at different time points to measure the level of Nell-1 protein, which can provide information indicative of the state of the disease. For example, if a patient's Nell-1 protein level shows a general trend of increasing over time, the patient is considered to be experiencing an improvement in the severity of the neurodegenerative disorder, or the treatment the patient is receiving is considered to be effective in treating the disorder. A lack of substantial change or a continuing decrease in the level of Nell-1 protein in the patient indicates a lack of change in the state of the neurodegenerative disorder and the ineffectiveness of the treatment administered to the patient.

[0051] Furthermore, the present inventors have devised a method for assessing (1) the relative risk of developing a neurodegenerative disorder, such as AD, MCI, or PD, or (2) the relative severity of a neurodegenerative disorder, such as AD, MCI, or PD, between two or more subjects based on the relative levels of Nell-1 protein in plasma, serum, or blood samples, where the lower the level of Nell-1 protein, the higher the risk or severity of the neurodegenerative disorder in that subject compared to others.

[0052] B. Sample Preparation for Protein Detection

[0053] Blood samples from subjects are suitable for the present invention and can be obtained by well-known methods and methods described in standard medical literature. In certain applications of the present invention, serum, plasma, or whole blood may be the preferred sample type. In other cases, whole blood samples may be used.

[0054] A blood sample is taken from a person to be tested, evaluated, or monitored for a neurodegenerative disorder using the method of the present invention. Collection of blood samples from subjects is carried out according to standard protocols commonly used in hospitals or clinics. An appropriate amount of blood is collected and can be stored according to standard procedures prior to further preparation.

[0055] Analysis of marker proteins (e.g., Nell-1 protein) found in patient samples according to the present invention can be performed using, for example, serum, plasma, or whole blood. Methods for preparing patient samples for protein extraction or quantitative detection are well known to those skilled in the art.

[0056] C. Determining the Levels of Marker Proteins

[0057] Proteins of any particular identity, such as Nell-1 protein, can be detected using a variety of immunological assays. In some embodiments, a sandwich assay can be used to capture a protein from a test sample using an antibody with specific binding affinity for the protein. The protein can then be detected using a labeled antibody with specific binding affinity for the protein. Such immunological assays can be performed using microfluidic devices such as microarray protein chips. A protein of interest (e.g., Nell-1 protein) can also be detected by gel electrophoresis (such as two-dimensional gel electrophoresis) and Western blot analysis using a specific antibody. Alternatively, a given protein (e.g., Nell-1 protein) can be detected using an appropriate antibody using standard immunohistochemistry techniques. Both monoclonal and polyclonal antibodies (including antibody fragments with desired binding specificity) can be used for the specific detection of polypeptides. Such antibodies and their binding fragments with specific binding affinity for a particular protein (e.g., Nell-1 protein) can be generated by known techniques.

[0058] In carrying out the present invention, other methods can also be used to measure the level of marker proteins. For example, various methods based on mass spectrometry have been developed to rapidly and accurately quantify target proteins even in a large number of samples. These methods use highly sophisticated instruments, such as triple quadrupole (Triple Q) instruments using multiple reaction monitoring (MRM), matrix-assisted laser desorption / ionization time-of-flight tandem mass spectrometers (MALDI TOF / TOF), ion trap instruments using selected ion monitoring (SIM) mode, and electrospray ionization (ESI)-based QTOP mass spectrometers. See, for example, Pan et al., J Proteome Res. 2009 February; 8(2):787-797.

[0059] III. Establishing standard controls

[0060] To establish a standard control for carrying out the methods of the present invention, a group of healthy individuals is first selected who do not have a neurodegenerative disorder as conventionally defined (e.g., AD, MCI, or PD) or who do not have an increased risk of developing said neurodegenerative disorder. These subjects are within appropriate parameters, if applicable, for the purpose of screening and / or monitoring neurodegenerative disorders using the methods of the present invention. Optionally, these subjects are of the same sex, similar age, or similar ethnic background as the test subject.

[0061] The health status of the selected subject is confirmed by well-established and routinely used methods, including, but not limited to, a general physical examination of the subject and a general review of their medical history.

[0062] Furthermore, the selected group of healthy subjects should be of a reasonable size so that the average amount or concentration of a marker protein (e.g., Nell-1 protein) in serum, plasma, or whole blood samples obtained from the group can reasonably be considered to represent the normal or average level in the general population of healthy people who do not have or are not at increased risk for a neurodegenerative disorder (e.g., AD, MCI, or PD, etc.). Preferably, the selected group includes at least 10, 20, 30, or 50 human subjects.

[0063] Once the average value of the marker protein is established based on the individual values ​​found in each subject of the selected healthy control group, this average, median, representative value, or profile is considered the standard control. The standard deviation is also determined during the same process. In some cases, separate standard controls may be established for separately defined groups with different characteristics, such as age, sex, or ethnic background.

[0064] IV. Monitoring and Treatment

[0065] A. Treatment with known therapeutic measures

[0066] In a related aspect, the present invention also aids in and enables treatment of a neurodegenerative disorder (e.g., AD, MCI, or PD) upon detecting the presence of the neurodegenerative disorder, an increased risk of developing a neurodegenerative disorder in the future, or worsening of the condition of the neurodegenerative disorder in a patient. In some embodiments, the method includes administering a therapeutic agent to the subject if the subject is determined to have an increased risk of AD, such as, for example, an antibody drug (e.g., lecanemab), an acetylcholinesterase inhibitor (e.g., donepezil, galantamine, rivastigmine), memantine, a glutamate receptor blocker, citalopram, fluoxetine, paroxetine, sertraline, trazodone, lorazepam, oxazepam, aripiprazole, clozapine, haloperidol, olanzapine, quetiapine, risperidone, ziprasidone, nortriptyline, a tricyclic antidepressant, a benzodiazepine, temazepam, zolpidem, zaleplon, chloral hydrate, coenzyme Q10, ubiquinone, coral calcium, ginkgo biloba, huperzine A, an omega-3 fatty acid, phosphatidylserine, or any combination thereof.

[0067] In some cases, after the diagnostic method steps described above and herein are completed, and optionally additional diagnostic tests are performed to corroborate the diagnosis (e.g., by CT scans or other brain imaging tests that show excessive loss of brain volume or by testing cognitive function that shows accelerated decline), if a patient is determined to already have AD or to be at significantly increased risk for developing AD in the future, an appropriate therapeutic or preventative regimen can be prescribed by a physician or other medical professional for the patient, with the aim of treating the patient and managing or alleviating ongoing symptoms or delaying the future onset of the disease. The U.S. Food and Drug Administration (FDA) has approved several cholinesterase inhibitors, including donepezil (Aricept™, the only cholinesterase inhibitor approved to treat all stages of AD, including moderate to severe), rivastigmine (Exelon™, approved to treat mild to moderate AD), galantamine (Razadyne™, for mild to moderate patients), and memantine (Namenda™). Donepezil is the only cholinesterase inhibitor approved to treat all stages of AD, including moderate to severe. Any one or more of these drugs can be prescribed to treat patients diagnosed with AD according to the methods of the present invention. In addition, Biogen's antibody drugs aducanumab and lecanemab were recently approved by the FDA. Another treatment possibility is the administration of trazodone, which is currently approved for use as an antidepressant and has been reported as an effective agent to improve AD symptoms.

[0068] Continuous monitoring, especially at increased frequency, may also be appropriate for patients who are considered to be at high or increased risk of developing a neurodegenerative disorder such as AD, MCI, or PD in the future but who have not yet shown clinical symptoms. For example, patients may undergo more frequently scheduled periodic testing (e.g., every 6 months, yearly, or biannually) to detect any accelerated changes in cognitive function. Suitable methods for such periodic monitoring include the General Practitioner Assessment of Cognition (GPCOG), Mini-Cog, the Eight-Item Informant Interview to Differentiate Aging and Dementia (AD8), and the Short Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE). Additionally, preventive treatment with trazodone may also be recommended.

[0069] In accordance with the methods of the present invention, for patients who already suffer from MCI or PD or who have been diagnosed as being at high risk for developing MCI or PD in the future, various medications can be used to treat these patients in both therapeutic and preventative settings. For example, patients diagnosed with or at risk for MCI can be treated with certain AD medications (e.g., aducanumab or lecanemab), or by discontinuing some of their currently administered medications (e.g., benzodiazepines used to treat conditions such as anxiety, seizures, and sleep disorders; anticholinergics that act on chemicals in the nervous system to treat various conditions; antihistamines used to manage allergy symptoms; opioids used for pain relief; and proton pump inhibitors used to treat reflux or gastroesophageal reflux disease (GERD)). Alternatively, medications to treat certain medical conditions known to contribute to the worsening of MCI symptoms (e.g., high blood pressure, depression, sleep apnea, etc.) can be administered. Additionally, patients may modify their lifestyle and social behavior to alleviate MCI symptoms, slow cognitive decline, or reduce the risk of MCI, including regular physical exercise, eating a diet low in fat and rich in fruits and vegetables, omega-3 fatty acid supplementation, engaging in a mentally and socially active lifestyle, and professionally delivered memory training and other cognitive training programs.

[0070] For patients diagnosed with or at risk for PD, the following medications may be appropriate: These include carbidopa-levodopa (including inhaled and continuous infusion forms), Duopa, dopamine agonists (e.g., pramipexole (Mirapex ER), rotigotine (Neupro), apomorphine (Apokyn)), monoamine oxidase B (MAO-B) inhibitors (e.g., selegiline (Zelapar), rasagiline (Azilect), safinamide (Xadago)), catechol-O-methyltransferase (COMT) inhibitors (e.g., entacapone (Comtan), opicapone (Ongentys), tolcapone (Tasmar)), anticholinergics, amantadine, adenosine receptor antagonists (A2A receptor antagonists, e.g., isladefylline (Nourianz)), and nuplazid (pimavanserin). Lifestyle and home remedies, including regular exercise and a healthy diet, can also be used to treat PD patients and individuals at risk for PD.

[0071] B. Targeting Nell-1 for Therapy

[0072] In addition to known therapeutic agents and uses for the treatment of neurodegenerative disorders such as AD, MCI, or PD, the discovery made by the inventors, i.e., the association between reduced levels of circulating Nell-1 protein and the presence, increased risk, or severity of neurodegenerative disorders such as AD, MCI, or PD, allows for novel and effective means of treating such neurodegenerative disorders, applicable in both prophylactic and therapeutic settings.

[0073] More specifically, the present invention provides the therapeutic use of Nell-1 protein or nucleic acid encoding said protein for treating neurodegenerative disorders, including AD, MCI, and PD, by enhancing the level of Nell-1 protein, particularly in the brain of a patient.For example, a composition comprising an effective amount of recombinant Nell-1 protein can be directly administered to a patient via an appropriate administration route and delivery system.In other cases, the rate of Nell-1 protein synthesis can be increased by regulating Nell-1 gene expression in cellular sources, focusing on an appropriate targeting site (e.g., the brain), using genetic engineering techniques (e.g., Nell-1 gene knock-in using a CRISPR-based system). Furthermore, treatment can be achieved by activating downstream signaling pathways of Nell-1 by stimulating the Nell-1-specific receptor Cntnap4 using one or more appropriate agonists, including agonist antibodies against Cntnap4, small molecules, or peptides (e.g., specific peptide fragments of Nell-1) (see, e.g., Li et al., J Bone M Res, 33(10), 1813-1825, 2018). Because Cntnap4 is primarily expressed in the brain, pharmaceutical compositions containing such agonists are preferably delivered using delivery carriers that can cross the blood-brain barrier and specifically target the brain.

[0074] The present invention provides pharmaceutical compositions comprising, or consisting essentially of, an effective amount of an active therapeutic agent (e.g., a Nell-1 protein or a nucleic acid encoding the Nell-1 protein, a Nell-1 receptor protein or a nucleic acid encoding the receptor, or an agonist / activator of the Nell-1 protein or its receptor) and one or more physiologically or pharmaceutically acceptable non-medicinal ingredients (excipients). The pharmaceutical compositions of the present invention are suitable for use in a variety of drug delivery systems. Formulations suitable for use in the present invention are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). For a brief review of methods of drug delivery, see Langer, Science 249:1527-1533 (1990).

[0075] The pharmaceutical compositions of the present invention can be administered by various routes, including systemic administration via intravenous, intramuscular, or subcutaneous injection, as well as local delivery via intracranial or intraperitoneal injection. One dosage form for administering the pharmaceutical composition involves intravenous administration of the nucleic acid of the present invention at a daily dose of about 1 to about 1000 μg, about 5 to about 500 μg, about 10 to about 250 μg, about 20 to about 100 μg, or about 25 to about 50 μg. Alternatively, the pharmaceutical composition can be administered intravenously at a daily dose of about 1 to about 1000 mg, about 5 to about 500 mg, about 10 to about 250 mg, about 20 to about 100 mg, or about 25 to about 50 mg of the recombinant protein or activator / agonist according to the present disclosure. Furthermore, the compositions may be formulated into daily, weekly, or monthly dosage forms for administration to a subject. A suitable dose may be administered as a one-time daily dose or as divided doses administered at appropriate intervals such as every 2 months, 3 months, 4 months, 5 months, 6 months, or more months (e.g., 12 months).

[0076] Polynucleotide sequences comprising RNA or any derivative or modified version thereof can be chemically synthesized according to methods known in the relevant art. RNA molecules can be modified with one or more nucleotide analogs and / or substitutions in the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization or binding ability, bioavailability, etc. Polynucleotides may also contain additional groups such as peptides (e.g., those that target host cell receptors), agents that cross cell membranes (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; WO 88 / 09810) or the blood-brain barrier (see, e.g., WO 89 / 10134), hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988, BioTechniques 6:958-976), or intercalators (see, e.g., Zon, 1988, Pharm. Res. 5:539-549).

[0077] In some embodiments, compositions containing active agents of the present invention, such as nucleic acids encoding Nell-1 proteins, are formulated as nanoparticle compositions, particularly compositions in the form of lipid nanoparticles (LNPs) containing an active agent (e.g., a nucleic acid, which may be in the form of DNA or RNA, including modified RNA). One or more lipids and other components can be used in the formulation. For example, LNPs may contain cationic lipids, neutral lipids, steroids, polymer-bound lipids, and an active agent (e.g., a nucleic acid, such as DNA or RNA, and chemically modified versions thereof). In some cases, LNPs may further contain at least one lipid or lipid-like substance other than a cationic lipid or a cationizable lipid or lipid-like substance, at least one polymer other than a cationic polymer, or a mixture thereof. A preferred mode of administration for DNA- or RNA-encapsulated LNP compositions is intravenous or intracranial administration, e.g., a preservative-free sterile dispersion containing the nucleic acid formulated in lipid nanoparticles in an aqueous cryoprotectant buffer for local or systemic injection. In some embodiments, the active agent-loaded LNP formulations of the present invention are used to facilitate effective passage across the blood-brain barrier, and optionally, to further enhance brain-specific delivery of the active agent, suitable affinity moieties that specifically target brain cells, such as binding partners for brain cell surface receptors, may be conjugated to lipid components present on the outer surface of the nanoparticles.

[0078] Furthermore, gene editing technology can be used in the therapeutic scheme of the present invention. For example, the CRISPR / Cas system has been adapted for use in targeted gene editing in eukaryotic cells. For example, see Ledford (2016), Nature 531 (7593):156-9. Additional gene editing systems that can be used to implement the present invention include recently developed technologies such as TALEN (transcription activator-like effector nuclease), ZFN (zinc finger nuclease), base editing, and homing endonucleases and meganucleases (MegN) that target and cut DNA sequences, and prime editing that generates RNA templates for gene modification.

[0079] As shown in the present disclosure, a lower-than-normal level of Nell-1 protein in a patient's blood, serum, or plasma sample indicates the presence or increased risk of a neurodegenerative disorder, such as AD, MCI, or PD, in the patient. Meanwhile, a lower level of Nell-1 protein corresponds to a worsening of the neurodegenerative disorder or a more advanced disease state. Therefore, to assess the effectiveness of a treatment regimen, the level of Nell-1 protein found in the patient's blood sample (e.g., a plasma or serum sample) is measured a reasonable period of time after administration of a therapeutic composition comprising an effective amount of an active agent of the present invention (e.g., a Nell-1 protein or a nucleic acid encoding said Nell-1 protein, a Nell-1 receptor protein or a nucleic acid encoding said receptor, or an agonist / activator of a Nell-1 protein or its receptor), preferably both before and after administration. Efficacy of the treatment is indicated by a finding that the level of Nell-1 protein in a blood, serum, or plasma sample from a patient after treatment is increased compared to the pre-treatment level found in the same type of blood sample from the patient, or that the level of Nell-1 protein in a blood, serum, or plasma sample from a patient after treatment is equal to or greater than the standard control value expected in the same type of blood sample from an average cognitively normal subject.

[0080] V. Kits and Equipment

[0081] The present invention provides compositions and kits for carrying out the methods described herein to assess the level of the marker protein Nell-1 in the serum, plasma, or whole blood of a subject, which can be used for a variety of purposes, including detecting or diagnosing the presence of a neurodegenerative disorder (e.g., AD, MCI, or PD), determining the risk of developing a neurodegenerative disorder, monitoring the progression of a neurodegenerative disorder in a patient, and assessing the effectiveness of treatments administered to patients diagnosed with and treated for a neurodegenerative disorder.

[0082] Kits for performing assays to determine the level of marker proteins typically include at least one antibody useful for specifically binding to a Nell-1 protein amino acid sequence. Optionally, the antibody is labeled with a detectable moiety. The antibody may be either a monoclonal or polyclonal antibody. In some cases, the kit may include at least two different antibodies: one antibody that specifically binds to a Nell-1 protein (a primary antibody) and another antibody for detecting the primary antibody (often a secondary antibody conjugated to a detectable moiety).

[0083] Typically, the kit also includes a suitable standard control. The standard control represents the average value of the marker protein Nell-1 in serum, plasma, or whole blood of healthy subjects who are not afflicted with a neurodegenerative disorder, such as AD, MCI, or PD, and who are not at increased risk of developing a neurodegenerative disorder. In some cases, such a standard control may be provided in the form of a set value. Furthermore, the kit of the present invention may provide instructions to guide the user in analyzing the test sample and assessing the presence or risk of a neurodegenerative disorder (e.g., AD, MCI, or PD), or assessing the status / severity / progression of the disorder in the test subject.

[0084] In a further aspect, the present invention may be in the form of a device or system including one or more such devices capable of performing all or some of the steps of the methods described herein. For example, in some cases, the device or system including a plurality of such devices performs the following steps after receiving a serum, plasma, or whole blood sample collected from a subject for the purpose of detecting a neurodegenerative disorder (such as AD, MCI, or PD), assessing the risk of developing the neurodegenerative disorder, or assessing the severity / status / progression of the disease: (a) measuring the amount or concentration of the marker protein Nell-1 in the sample; (b) comparing the measured amount / concentration with a standard control value; and (c) providing a result indicating whether the subject has a neurodegenerative disorder (e.g., AD, MCI, or PD), whether they have an increased risk of developing the neurodegenerative disorder, or whether they have a higher relative risk of developing the neurodegenerative disorder in the future compared to other test subjects. In other cases, the device or system of the present invention performs the tasks of steps (b) and (c) after step (a) is performed and the amount or concentration obtained in (a) is input into the device. Preferably, the device or system is partially or fully automated.

[0085] Example

[0086] The following examples are offered by way of illustration only and are not intended to be limiting. A person of ordinary skill in the art will readily recognize that various non-critical parameters can be changed or modified to yield essentially the same or similar results.

[0087] Introduction

[0088] Neurological disorders include a wide range of diseases of the nervous system. For example, Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline. AD is the leading cause of dementia and the seventh leading cause of death worldwide. As of 2020, more than 55 million people worldwide suffered from dementia, and due to the accelerating aging of the global population, the patient population is expected to reach 139 million by 2050 (Gauthier et al., World Alzheimer Report 2021: Journey through the diagnosis of dementia. London, England: Alzheimer's Disease International). Unfortunately, there are currently no effective treatments that can halt or reverse the progression of AD.

[0089] Limited diagnostic tools for AD hinder the development of treatments. The progression of AD involves an extensive preclinical period during which pathological changes occur in the brain without the appearance of cognitive impairment. Identifying and correcting disease-causing factors at an early stage is desirable for effective treatment. However, current diagnostic methods for AD (e.g., clinical assessment of cognitive function, brain imaging, and detection of amyloid beta and tau proteins in cerebrospinal fluid) are inconvenient and expensive, and often only detect AD in its late stages. Therefore, there is an urgent need for new diagnostic biomarkers that can detect pathological changes in the brain at an early stage and are accessible to the general public.

[0090] Several plasma proteins have been shown to exert regulatory effects on brain functions, such as neurogenesis and microglial activation, either directly through the blood-brain barrier or indirectly by modulating the activity of infiltrating immune cells. Furthermore, many plasma proteins are dysregulated in the blood of patients with neurological diseases, such as AD. Collectively, dysregulated plasma proteins may serve as biomarkers for monitoring disease progression or therapeutic targets.

[0091] Here, Nell-1 (brain-derived growth factor-like protein) has been identified as a promising diagnostic biomarker and therapeutic target for neurological diseases. Decreased plasma Nell-1 protein levels can be used to distinguish patients with neurological diseases, such as AD, mild cognitive impairment (MCI), and Parkinson's disease (PD), from cognitively normal individuals. Plasma Nell-1 levels can also be used to monitor neurodegeneration and other associated pathological changes in the brain. Furthermore, elevated plasma Nell-1 levels have beneficial effects on the brain, including suppressing neurodegeneration, enhancing synaptic function, and promoting synaptic plasticity and neurogenesis. In conclusion, Nell-1 has been demonstrated not only as an effective blood biomarker for monitoring and classifying neurological diseases, but also as a therapeutic target for treating such diseases.

[0092] Materials and Methods

[0093] Targeted recruitment

[0094] Hong Kong Chinese over 60 years of age were enrolled from the Special Outpatient Department of the Prince of Wales Hospital, The Chinese University of Hong Kong (n = 119, n = 109, n = 152, and n = 39 for cognitively normal (CN) subjects, MCI patients, AD patients, and PD patients, respectively). Clinical diagnoses were performed according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) of the American Psychiatric Association. This study was approved by the Prince of Wales Hospital, The Chinese University of Hong Kong, and the Hong Kong University of Science and Technology. All participants provided written informed consent for both study participation and sample collection.

[0095] Plasma protein detection

[0096] Blood samples were collected into EDTA tubes using standard protocols. Plasma was prepared by centrifugation and stored at -80°C until use. Plasma levels of Nell-1 were measured using the Olink Oncology III panel. Plasma levels of neurofilament light polypeptide (NfL) and phosphorylated tau-181 (pTau181) were measured using the Simoa Neurology 4-Plex E Advantage Kit and P-Tau 181 Advantage V2 Kit, respectively.

[0097] Whole genome sequencing

[0098] Participant DNA samples were submitted to Novogene for library construction and whole-genome sequencing (WGS). Samples were sequenced using an Illumina Hiseq X, with an average sequencing depth of 5x. Genotype results, stored in VCF files, were used for principal component analysis. The top five principal components were generated using PLINK software.

[0099] Analysis of the association between plasma proteins and disease phenotypes

[0100] Plasma Nell-1 levels were transformed by rank-based inverse normalization using the RankNorm function in the RNOmni package in R. Dysregulation of plasma Nell-1 levels in patients with neurological diseases such as MCI, AD, and PD was determined by comparing normalized plasma Nell-1 levels with disease phenotypes, adjusting for age and sex, compared with controls. The following linear model was used (βi is a weighting coefficient, ε is the intercept):

[0101] Normalized plasma Nell-1 level = β1 × disease phenotype + β2 × age + β3 × sex + ε

[0102] The relationship between plasma Nell-1 levels and neurodegeneration-related endophenotypes, such as plasma NfL and plasma pTau181 levels, was determined using the association between normalized plasma Nell-1 levels and endophenotypes after adjusting for age and sex. The following linear model was used:

[0103] Normalized plasma Nell-1 level = β1 × neurodegeneration-related endophenotype + β2 × age + β3 × sex + ε

[0104] Evaluation of prediction accuracy

[0105] Receiver operating characteristic (ROC) curves and areas under the curve (AUC) for the predictive models for MCI and AD risk prediction were generated using GraphPad Prism (version 8.2.0). The accuracy of the predictive models was indicated by the AUC values.

[0106] One-sample Mendelian randomization analysis

[0107] Associations between plasma Nell-1 levels and all variants at the NELL1 locus (±1 Mb) were analyzed using linear regression adjusted for age, sex, disease phenotype, and the first five principal components of ancestry (n = 108 and n = 147 for CN participants and AD patients from the Hong Kong Chinese AD cohort, respectively). Association tests were performed using PLINK (version 1.9). A minor allele frequency >1% and a Hardy-Weinberg P value of 5 × 10 were considered significant. -6 Genetic variants exceeding 0.01 were selected for analysis. Given the limited sample size, genetic associations were considered significant when P < 0.01. LD-based clumping was performed to retain only the most significant variants (i.e., variants with the smallest P value) in each cluster and exclude variants in linkage disequilibrium (R 2Finally, the following 20 genetic variants were deemed suitable for use as instrumental variables for Mendelian randomization (MR) analysis: rs10741830, rs10833529, rs10833718, rs11025266, rs11026285, rs12795698, rs138024111, rs140262672, rs147617279, rs148361725, rs17840362, rs3045385, rs4923193, rs61155013, rs7109477, rs7112107, rs75540817, rs77814312, rs7938616, and rs867221555.

[0108] MR allows for the assessment of causal relationships between exposures (e.g., plasma Nell-1 levels) and outcomes (e.g., neurodegeneration-related endophenotypes) using their association with instrumental variables. Here, one-sample MR analysis was performed using two-stage least squares regression. Briefly, predicted plasma Nell-1 levels for each subject were calculated based on the genotype, which was an instrumental variable. These predicted values ​​were then regressed against neurodegeneration-related endophenotypes (e.g., plasma NfL levels and plasma pTau181 levels). The significance threshold was set at P<0.05.

[0109] Rat primary hippocampal neuron culture

[0110] Sprague-Dawley rat embryos were sacrificed on embryonic day 18, and the hippocampi were isolated and dissociated using trypsin. Hippocampal cells were plated at 0.3 × 10 per 18 mm coverslip coated with 1 mg / mL poly-D-lysine. 5Cells were seeded at a density of 1 / 3 cells / mL. Cultured cells were maintained in Neurobasal medium (Invitrogen) supplemented with 2% B27 (Invitrogen) and 0.5 mM L-glutamine and incubated at 37°C in a humidified atmosphere containing 5% CO2. To investigate the role of Nell-1 in excitatory synapse formation, cultured hippocampal cells were treated with recombinant human Nell-1 (2 μg / mL, R&D Systems) or DPBS at 12–14 days in vitro (DIV).

[0111] Immunostaining and quantification of cultured rat primary hippocampal neurons

[0112] After 48 hours of treatment, cells were fixed with 4% paraformaldehyde / 4% sucrose (weight / volume) and immunostained with VGluT1 (AB5905, Sigma-Aldrich) and PSD-95 (ab18258, Abcam) antibodies. Cultures were then counterstained with DAPI and mounted in ProLong Diamond Antifade Mountant (P36961, Invitrogen). Images were acquired using a Leica TCS SP8 confocal system. To examine changes in excitatory synapses in cultured hippocampal cells, VGluT1-positive PSD-95 clusters were quantified using ImageJ.

[0113] mouse

[0114] All experiments involving mice were approved by the Animal Ethics Committee of the Hong Kong University of Science and Technology (HKUST) and conducted in accordance with the guidelines of the HKUST Animal Care Facility. All mice were housed under a 12-h light / dark cycle in the HKUST Animal and Plant Care Facility. AAPP / PS1 transgenic mice were obtained from the Jackson Laboratory. Mice were randomly assigned to experimental conditions.

[0115] In vivo experiments in mice

[0116] Miniosmotic pumps (Model 1004, Alzet) were prepared with recombinant mouse NELL1 protein (7109-NL, R&D Systems, 189 ng per pump, 0.21 ng / h) or artificial cerebrospinal fluid (ASF) as a control. The pump cannula was then implanted into the ventricle for intraventricular delivery. Mice were sacrificed 7 days after the start of treatment.

[0117] electrophysiology

[0118] Mice were dissected immediately after sacrifice, and their brains were immersed in ice-cold artificial cerebrospinal fluid oxygenated with 95% O2 / 5% CO2. Subsequently, the brains were sliced ​​into 300 μm sections using a vibrating blade microtome (VT1000S, Leica). Brain slices were placed on a MED-P 210A probe (Panasonic International), and electrodes were positioned in the hippocampal region. Excitatory postsynaptic field potentials (fEPSPs) were recorded from the dendritic cell layer of hippocampal CA1 neurons. The baseline stimulation intensity was selected to elicit 30%–40% of the maximum fEPSP response. Long-term potentiation (LTP) was induced by three high-frequency stimulations (100 Hz, 1 s) administered at 30 s intervals. After tetanic stimulation, responses were recorded for 1 h. LTP was quantified as the change in the mean slope of the fEPSP over 1 h after LTP induction.

[0119] Immunostaining of mouse brain

[0120] Mice were dissected, and their brains were fixed in 4% paraformaldehyde at 4°C for 24 hours. Fixed brains were sliced ​​into 30 μm sections using a vibrating blade microtome (VT1200S, Leica). The sections were washed with DPBS and blocked with a blocking solution containing 4% goat serum, 1% BSA, 0.4% Triton X-100, and DPBS for 1 hour at room temperature. Subsequently, the sections were labeled with DCX antibody (AB2253, Sigma-Aldrich) overnight at 4°C. The next day, the sections were labeled with a fluorescent dye-conjugated anti-guinea pig secondary antibody for 2 hours at room temperature. The sections were then labeled with a nuclear staining dye (DAPI) and mounted on slides. Images of the sections were captured using a ZEISS LSM 980 equipped with Airyscan 2.

[0121] Statistical analysis and data visualization

[0122] The investigators who performed protein detection were blinded to the phenotype of the human subjects. The significance of the association analyses was assessed by linear regression adjusted for age and sex. The significance level was set at P<0.05. Statistical plots were generated using the ggplot function in the ggplot2 package in R or GraphPad Prism (version 8.2.0).

[0123] Example I: Assessing AD risk using Nell-1 protein levels in blood

[0124] Nell-1 protein levels were measured in plasma samples collected from a Hong Kong Chinese cohort (n = 119 cognitively normal [CN] subjects and 152 Alzheimer's disease [AD] patients). Plasma Nell-1 protein levels were significantly decreased in AD patients compared with CN subjects (β = -0.158, P < 0.05; Figure 1a). Therefore, plasma Nell-1 protein levels could be used to distinguish AD patients from CN subjects with 64.38% accuracy (Figure 1b). Furthermore, lower plasma Nell-1 protein levels may indicate a higher risk of developing AD. Taken together, blood Nell-1 protein levels may be useful for assessing the risk of developing AD.

[0125] Example II: Assessing MCI risk using Nell-1 protein levels in blood

[0126] Nell-1 protein levels were measured in plasma samples collected from a Hong Kong Chinese cohort (n = 119 cognitively normal [CN] subjects and n = 109 mild cognitive impairment [MCI] patients). Plasma Nell-1 protein levels were significantly decreased in MCI patients compared with CN subjects (β = -0.187, P < 0.05; Figure 2a). Therefore, plasma Nell-1 protein levels could be used to distinguish MCI patients from CN subjects with 65.25% accuracy (Figure 2b). Furthermore, lower plasma Nell-1 protein levels may indicate a higher risk of developing MCI. Taken together, blood Nell-1 protein levels may be useful for assessing the risk of developing MCI.

[0127] Example III: Assessing PD risk using Nell-1 protein levels in blood

[0128] Nell-1 protein levels were measured in plasma samples collected from a Hong Kong Chinese cohort (n = 119 cognitively normal [CN] subjects and n = 39 Parkinson's disease [PD] patients). Plasma Nell-1 protein levels were significantly decreased in PD patients compared with CN subjects (β = -0.370, P < 0.001; Figure 3a). Therefore, plasma Nell-1 protein levels could be used to distinguish PD patients from CN subjects with 67.27% accuracy (Figure 3b). Furthermore, lower plasma Nell-1 protein levels may indicate a higher risk of developing PD. Taken together, blood Nell-1 protein levels may be useful for assessing the risk of developing PD.

[0129] Example IV: Using Nell-1 protein levels in the blood to assess the level of neurodegeneration, AD progression, and associated pathological changes in the brain

[0130] Nell-1 protein levels were measured in plasma samples collected from the Hong Kong Chinese cohort. Plasma Nell-1 levels correlated with neurodegeneration as indicated by plasma NfL levels (β = -0.005, R 2 = 0.1037, P < 0.0001, Fig. 4a) and AD progression indicated by pTau181 levels in plasma (β = -0.043, R 2 = 0.0808, P = 0.0076, Figure 4b). Furthermore, plasma Nell-1 levels were positively associated with cognitive function as measured by the MoCA score (β = 0.012, R 2 = 0.0337, P = 0.0098, Figure 4c), and was negatively associated with aging (β = -0.014, R 2 =0.0964, P<0.0001, FIG. 4d). Taken together, Nell-1 protein levels in the blood may be useful for monitoring the progression of AD-related pathological changes in the brain.

[0131] Example V: Inhibition of neurodegeneration in the brain by increasing Nell-1 protein levels or activity

[0132] Nell-1 protein levels were measured in plasma samples collected from a Hong Kong Chinese cohort. Whole genome sequencing was also performed on DNA samples collected from the Hong Kong Chinese cohort. Genetic variants regulating plasma Nell-1 protein levels were identified (P<0.01, Figure 5a). Genetic variants were used as instrumental variables to analyze the causal relationship between plasma Nell-1 levels and AD-related endophenotypes via Mendelian randomization (MR) analysis. MR analysis demonstrated that elevated plasma Nell-1 levels had a causal effect on suppressing neurodegeneration, as indicated by reduced plasma NfL levels (β=-0.278, P=0.0019, Figure 5b). This indicates that Nell-1 protein has a protective effect against neurodegeneration.

[0133] Example VI: Improving neuronal function in the brain by increasing Nell-1 protein levels or activity

[0134] Nell-1 protein was administered to primary cultured rat hippocampal neurons. Nell-1 treatment resulted in a significant increase in the expression of presynaptic and postsynaptic proteins in neuronal processes (P<0.001, Figures 6a-c). Furthermore, Nell-1 treatment significantly increased the number of excitatory synapses, as indicated by overlapping presynaptic and postsynaptic proteins (P<0.001, Figures 6a-c). Collectively, administration of Nell-1 to neurons has beneficial effects on neuronal and synaptic function.

[0135] Example VII: Improving synaptic plasticity in the brain by increasing Nell-1 protein levels or activity

[0136] Nell-1 protein was administered intracerebroventricularly to 11-month-old wild-type (WT) mice or APP / PS1 mice (i.e., a transgenic mouse model of AD) for 7 days. Nell-1 treatment reversed the impairment of hippocampal long-term potentiation (LTP) in APP / PS1 mice (P<0.05, Fig. 7a, b). LTP is a form of synaptic plasticity associated with memory, involving long-term enhancement of synaptic plasticity. In summary, in vivo treatment with Nell-1 has beneficial effects on synaptic plasticity.

[0137] Example VIII: Improving neurogenesis in the brain by increasing Nell-1 protein levels or activity

[0138] Nell-1 protein was administered intracerebroventricularly to 11-month-old WT mice for 7 days. Nell-1 treatment resulted in an increase in the density of immature neurons in the dentate gyrus of the hippocampus (P<0.05, Figures 8a-c). This finding suggests that Nell-1 treatment has a beneficial effect on neurogenesis.

[0139] All patents, patent applications, and other publications cited herein (including GenBank accession numbers and their equivalents) are hereby incorporated by reference in their entirety for all purposes.

Claims

1. (1) comparing the level of Nell-1 protein in the subject's plasma, serum, or whole blood with a standard control level of Nell-1 protein found in the plasma, serum, or whole blood of an average healthy subject who is not afflicted with or at increased risk for a neurodegenerative disorder; and (2) detecting a decrease in the level of Nell-1 protein in the subject's plasma, serum, or whole blood from the standard control level, and determining that the subject is suffering from or at increased risk of the neurodegenerative disorder; or (3) detecting that the level of Nell-1 protein in the subject's plasma, serum, or whole blood is not decreased from the standard control level, and determining that the subject is not suffering from or at increased risk of the neurodegenerative disorder.

10. A method for diagnosing, monitoring, or assessing the risk of a neurodegenerative disorder in a subject, comprising:

2. The method of claim 1, further comprising measuring the level of Nell-1 protein in plasma, serum, or whole blood prior to step (1).

3. 3. The method of claim 2, further comprising obtaining a sample of plasma, serum, or whole blood from the subject prior to said measuring step.

4. 2. The method of claim 1, further comprising, after step (3), comparing the level of Nell-1 protein in the plasma, serum, or whole blood of the subject measured at a later time point with the level of Nell-1 protein in the plasma, serum, or whole blood of the subject measured at step (1), wherein a higher level of Nell-1 protein in the plasma, serum, or whole blood measured at the later time point indicates an improvement in the neurodegenerative disorder, and a lower level of Nell-1 protein in the plasma, serum, or whole blood measured at the later time point indicates a worsening of the neurodegenerative disorder.

5. 5. The method of claim 4, wherein between step (3) and the later time point, the subject has been administered a therapeutic agent intended to treat the neurodegenerative disorder.

6. The method of any one of claims 1 to 5, wherein the neurodegenerative disorder is Alzheimer's disease (AD).

7. The method according to any one of claims 1 to 5, wherein the neurodegenerative disorder is mild cognitive impairment (MCI) or Parkinson's disease (PD).

8. 1. A method of assessing the severity or risk of a neurodegenerative disorder in two subjects, comprising: (i) comparing the level of Nell-1 protein in the plasma, serum, or whole blood of a first subject with the level of Nell-1 protein in the plasma, serum, or whole blood of a second subject; (ii) detecting a level of Nell-1 protein in the plasma, serum, or whole blood of the second subject that is lower than the level of Nell-1 protein in the plasma, serum, or whole blood of the first subject; and (iii) determining that the second subject has a less severe or lower risk of the neurodegenerative disorder than the first subject. A method comprising:

9. 9. The method of claim 8, wherein the neurodegenerative disorder is Alzheimer's disease (AD).

10. 9. The method of claim 8, wherein the neurodegenerative disorder is mild cognitive impairment (MCI) or Parkinson's disease (PD).

11. The method of claim 8 or claim 9, further comprising measuring the level of Nell-1 protein in plasma, serum, or whole blood prior to step (i).

12. 12. The method of claim 11, further comprising obtaining a sample of plasma, serum, or whole blood from the subject prior to said measuring step.

13. a first reagent capable of determining the level of Nell-1 protein in the plasma, serum, or whole blood of a subject, and optionally a second reagent capable of determining the level of phosphorylated Tau-181 (pTau181) or neurofilament light polypeptide (NfL) in the plasma, serum, or whole blood of said subject; A kit for diagnosing a neurodegenerative disorder or assessing the severity or risk of a neurodegenerative disorder in a subject.

14. 13. The kit of claim 12, comprising reagents capable of determining levels of pTau181 and NfL in the plasma, serum, or whole blood of the subject.

15. 13. The kit of claim 12, further comprising a standard control that reflects the level of Nell-1 protein found in the plasma, serum, or whole blood of an average healthy subject who is not afflicted with and not at increased risk for a neurodegenerative disorder.

16. The kit according to any one of claims 12 to 15, wherein the neurodegenerative disorder is Alzheimer's disease (AD), mild cognitive impairment (MCI), or Parkinson's disease (PD).

17. (1) administering to a subject in need thereof an effective amount of a Nell-1 enhancer that enhances the expression or activity of a Nell-1 protein, or (2) administering to a subject in need thereof an effective amount of a Nell-1 receptor enhancer that enhances the expression or activity of a Nell-1 receptor; A method of treating or reducing the risk of a neurodegenerative disorder.

18. 18. The method of claim 17, wherein the Nell-1 enhancer is a Nell-1 protein or a nucleic acid encoding a Nell-1 protein.

19. 18. The method of claim 17, wherein the Nell-1 enhancer is a Nell-1 receptor, a nucleic acid encoding the Nell-1 receptor, or an agonist of the Nell-1 receptor.

20. 18. The method of claim 17, wherein said administering comprises brain-targeted delivery of said Nell-1 enhancer or said Nell-1 receptor enhancer.

21. 21. The method of claim 19 or claim 20, wherein the nucleic acid encoding the Nell-1 protein or the Nell-1 receptor is formulated in a lipid nanoparticle composition for delivery.

22. 21. The method of claim 19 or claim 20, wherein the nucleic acid encoding the Nell-1 protein or Nell-1 receptor is introduced by gene editing.

23. The method of any one of claims 17 to 22, further comprising measuring the level of Nell-1 protein in the plasma, serum, or whole blood of the subject before and / or after the administering step.

24. 24. The method of claim 23, further comprising obtaining a sample of plasma, serum, or whole blood from the subject prior to one or more of said measuring steps.

25. The method of any one of claims 17 to 24, wherein the neurodegenerative disorder is Alzheimer's disease (AD), mild cognitive impairment (MCI), or Parkinson's disease (PD).

26. 1. A method for assessing the effectiveness of a therapeutic agent for treating a neurodegenerative disorder in a subject, comprising: (1) comparing the level of Nell-1 protein in the plasma, serum, or whole blood of the subject before and after administering the therapeutic agent to the subject; (2) detecting an increase in the level of Nell-1 protein in the plasma, serum, or whole blood of the subject after administration of the therapeutic agent; and (3) determining that the therapeutic agent is effective in treating the neurodegenerative disorder; A method comprising:

27. 27. The method of claim 26, wherein the neurodegenerative disorder is Alzheimer's disease (AD), mild cognitive impairment (MCI), or Parkinson's disease (PD).

28. The method of claim 26 or 27, further comprising, before step (1), measuring the level of Nell-1 protein in plasma, serum, or whole blood before and after administration.

29. 30. The method of claim 28, further comprising obtaining plasma, serum, or whole blood samples from the subject before and after administration, prior to said measuring step.

30. The method of any one of claims 1 to 12 and claims 17 to 29, wherein the subject is of Chinese descent.

Images