Compositions and Articles Comprising ACTIVITY DEPENDENT NEUROTROPHIC FACTOR (ADNF) POLYPEPTIDES

Abstract

Compositions and articles comprising ADNF polypeptides are provided. Accordingly, articles of manufacture are provided that comprise ADNF polypeptides and SIRT1 activators as active ingredients. Methods of treating diseases that can benefit from the articles of manufacture are also provided. [Representative figure] Figure 1B

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Patent Application No. 63 / 165,801, filed March 25, 2021, and U.S. Patent Application No. 63 / 178,026, filed April 22, 2021, the contents of which are incorporated by reference in their entireties herein.

[0002] Description of sequence listing An ASCII file submitted contemporaneously with the filing of this application, entitled 91463SequenceListing.txt, created on March 24, 2022, and containing 23,149 bytes, is hereby incorporated by reference.

[0003] Technical Field and Background Art The present invention, in some aspects, relates to compositions and articles comprising Activity Dependent Neurotrophic Factor (ADNF) polypeptides.

[0004] Activity-Dependent Neuroprotective Protein (ADNP or ADNF III) is essential for brain formation and function. ADNP has been shown to function in important cellular activities including embryogenesis, autophagy, dendritic spine plasticity, axonal transport, alternative RNA splicing, wnt signaling, autism-related protein translation, and chromatin remodeling. De novo mutations in ADNP lead to the autistic ADNP syndrome. 18、19 Somatic ADNP mutations may cause Alzheimer's Disease (AD) tauopathy 15 Furthermore, decreased circulating ADNP expression was associated with increased inflammation. 20 and cognitive decline 21ADNP is found in the nucleus as part of the SWItch / sucrose nonfermenting (SWI / SNF) complex, which constitutes a major part of the chromatin remodeling complex. 33 In mature neurons, ADNP associates with microtubules through interactions with the microtubule end-binding proteins, EB1 and EB3. 10 , found in the cytoplasm 9 Second, EB1 / EB3 interaction with ADNP mediates dendritic spine formation. 10、11 , axonal transport 12 , Enhancement of tau-microtubule binding 13~15 , and protection against tau hyperphosphorylation / tauopathy 14~16 is associated with.

[0005] ADNP polypeptides, including a proline-rich 8-amino acid polypeptide known as NAP [NAPVSIPQ (SEQ ID NO:2), also known as Davunetide or CP201], and their use in neuroprotection and treatment of several disorders are described in PCT International Publication Nos. WO 1 / 92333, WO 98 / 35042, WO 00 / 27875, WO 00 / 53217, WO 01 / 12654, WO 2004 / 080957, WO 2006 / 099739, WO 2007 / 096859, WO 2008 / 080957, WO 2009 / 080959, WO 2010 / 080959, WO 2011 / 080959, WO 2012 / 080959, WO 2013 / 080959, WO 2014 / 080959, WO 2015 / 080959, WO 2016 / 080959, WO 2017 / 080959, WO 2018 ...5 / 080959, WO 2016 / 080959, WO 2 Nos. 4,483, 5,767,240, 6,174,862, and 6,613,740, each of which is incorporated herein by reference in its entirety.

[0006] Sirtuin 1 (SIRT1) has been positively linked to aging. SIRT1 deacetylates many substrates, including histones, and as such is involved in chromatin remodeling, enhancing synaptic plasticity, maintaining genomic stability, suppressing inflammation, and protecting against neurodegeneration. 24、59 SIRT1 deficiency leads to accumulation of hyperphosphorylated tau, tauopathy, and AD 23 High SIRT1 expression has been shown to protect elderly people from dementia 22 . Summary of the Invention

[0007] According to one aspect of some embodiments of the present invention, there is provided an article of manufacture comprising as active ingredients an ADNF polypeptide, the ADNF polypeptide having neurotrophic / neuroprotective activity in an in vitro cortical neuronal culture assay, and a SIRT1 activator.

[0008] According to an aspect of some embodiments of the present invention there is provided a method of treating a disease in a subject in need of treatment selected from the group consisting of ADNP syndrome, Dravet syndrome, Fragile X syndrome, SYNGAP1 associated intellectual disability, Phelan-McDermid syndrome, GRIN disorder, CHD8 associated disorder, DYRK1A syndrome, POGZ syndrome, FOXP1 syndrome, SLC5A1 associated disorder, Coffin-Siris syndrome, ARID1B associated syndrome, KMT5B syndrome, PTEN autism syndrome, Rett syndrome, Okihiro syndrome plus developmental delay, Angelman syndrome, Noonan syndrome, Kleefstra syndrome, and Smith-Maginnis syndrome, comprising administering to the subject a therapeutically effective amount of a SIRT1 activator, thereby treating the disease in the subject.

[0009] According to some embodiments of the invention, the SIRT1 activator is a small molecule.

[0010] According to some embodiments of the invention, the SIRT1 activator is selected from the group consisting of resveratrol, quercetin, butein, beverine, curcumin, fisetin, honokiol, YK3-237, SRT1720, SRT1460, SRT2183, STAC-5, STAC-9, STAC-10, BML-278, and piceatannol, or an analogue or derivative thereof.

[0011] According to some embodiments of the invention, the SIRT1 activator is resveratrol or an analogue or derivative thereof.

[0012] According to some embodiments of the invention, the SIRT1 activator is NAD+ or an analog or derivative thereof.

[0013] According to some embodiments of the invention, the SIRT1 activator is Nicotinamide Riboside (NR).

[0014] According to an aspect of some embodiments of the present invention, there is provided an article of manufacture comprising as active ingredients an ADNF polypeptide, the ADNF polypeptide having neurotrophic / neuroprotective activity in an in vitro cortical neuronal culture assay, and an anti-aging agent, wherein the anti-aging agent is not an antioxidant.

[0015] According to an aspect of some embodiments of the present invention there is provided a method of treating a disease in a subject in need of treatment selected from the group consisting of ADNP syndrome, Dravet syndrome, Fragile X syndrome, SYNGAP1-associated intellectual disability, Phelan-McDermid syndrome, GRIN disorder, CHD8-associated disorder, DYRK1A syndrome, POGZ syndrome, FOXP1 syndrome, SLC5A1-associated disorder, Coffin-Siris syndrome, ARID1B-associated syndrome, KMT5B syndrome, PTEN autism syndrome, Rett syndrome, Okihiro syndrome plus developmental delay, Angelman syndrome, Noonan syndrome, Kleefstra syndrome, and Smith-Maginnis syndrome, comprising administering to the subject a therapeutically effective amount of an anti-aging agent, thereby treating the disease in the subject.

[0016] According to some embodiments of the present invention, the anti-aging agent is not an antioxidant.

[0017] According to some embodiments of the invention, the anti-aging agent is a SIRT1 activator.

[0018] According to some embodiments of the present invention, the anti-aging agent is selected from the group consisting of rapamycin, metformin, melatonin, carnosine, nicotinamide mononucleotide, δ-sleep inducing peptide and low molecular weight klotho enhancer, or analogs or derivatives thereof.

[0019] According to some embodiments of the present invention, the anti-aging agent comprises a calorie restricted diet.

[0020] According to one aspect of some embodiments of the present invention, there is provided an article of manufacture comprising as active ingredients an ADNF polypeptide, the ADNF polypeptide having neurotrophic / neuroprotective activity in an in vitro cortical neuronal culture assay, and an immunomodulatory agent selected from the group consisting of a chemokine receptor modulator, an immune checkpoint modulator, and a cytokine, wherein the cytokine is not IFNβ.

[0021] According to an aspect of some embodiments of the present invention there is provided a method of treating a disease selected from the group consisting of ADNP syndrome, Dravet syndrome, Fragile X syndrome, SYNGAP1 associated intellectual disability, Phelan-McDermid syndrome, GRIN disorder, CHD8 associated disorder, DYRK1A syndrome, POGZ syndrome, FOXP1 syndrome, SLC5A1 associated disorder, Coffin-Siris syndrome, ARID1B associated syndrome, KMT5B syndrome, PTEN autism syndrome, Rett syndrome, Okihiro syndrome plus developmental delay, Angelman syndrome, Noonan syndrome, Kleefstra syndrome, and Smith-Maginnis syndrome in a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of an immunomodulatory agent selected from the group consisting of a chemokine receptor modulator, an immune checkpoint modulator, and a cytokine, thereby treating the disease in the subject.

[0022] According to some embodiments of the invention, the cytokine is not IFNβ.

[0023] According to some embodiments of the invention, the chemokine receptor is selected from CCR5 and CXCR4.

[0024] According to some embodiments of the invention, the modulator is an inhibitor.

[0025] According to some embodiments of the invention, the chemokine receptor modulator is selected from the group consisting of maraviroc, leronlimab, aplaviroc, vicriviroc, plerixafor, mavorixafor, BL-8040, and TGO-0054, or an analogue or derivative thereof.

[0026] According to some embodiments of the invention, the chemokine receptor modulator is selected from the group consisting of maraviroc and plerixafor, or an analogue or derivative thereof.

[0027] According to some embodiments, the cytokine is selected from the group consisting of IL-6, IL-10, and TNFα.

[0028] According to an aspect of some embodiments of the present invention there is provided an article of manufacture comprising as active ingredients an ADNF polypeptide, the ADNF polypeptide having neurotrophic / neuroprotective activity in an in vitro cortical neuronal culture assay, and bumetanide or an analog or derivative thereof.

[0029] According to one aspect of some embodiments of the present invention there is provided an article of manufacture comprising as active ingredients an ADNF polypeptide, the ADNF polypeptide having neurotrophic / neuroprotective activity in an in vitro cortical neuronal culture assay, and a cannabinoid.

[0030] According to some embodiments of the present invention, the cannabinoid is selected from the group consisting of THC and CBD.

[0031] According to an aspect of some embodiments of the present invention, there is provided an article of manufacture comprising as active ingredients an ADNF polypeptide, the ADNF polypeptide having neurotrophic / neuroprotective activity in an in vitro cortical neuronal culture assay, and ketamine, or an analog or derivative thereof.

[0032] According to some embodiments of the invention, the method further comprises administering to the subject a therapeutically effective amount of an ADNF polypeptide, the ADNF polypeptide having neurotrophic / neuroprotective activity in an in vitro cortical neuronal culture assay.

[0033] According to some embodiments of the invention, the ADNF polypeptide is capable of binding to EB1 and / or EB3.

[0034] According to some embodiments of the invention, the ADNF polypeptide is an ADNF III polypeptide.

[0035] According to some embodiments of the invention, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-22.

[0036] According to some embodiments of the invention, the polypeptide comprises SEQ ID NO:2.

[0037] According to some embodiments of the invention, the polypeptide has the formula 1 ) x -Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-(R 2 ) y (SEQ ID NO: 49), or an analog thereof, wherein R 1 R is an amino acid sequence comprising from 1 to about 40 amino acids, each amino acid being independently selected from the group consisting of naturally occurring amino acids and amino acid analogs. 2 is an amino acid sequence comprising from 1 to about 40 amino acids, where each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs, and x and y are independently selected and equal to 0 or 1.

[0038] According to some embodiments of the invention, the ADNF polypeptide is an ADNF I polypeptide.

[0039] According to some embodiments of the invention, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:24-48.

[0040] According to some embodiments of the invention, the polypeptide comprises SEQ ID NO:24.

[0041] According to some embodiments of the invention, the polypeptide has the formula 1 ) x -Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-(R 2 ) y (SEQ ID NO:50), or an analog thereof, wherein R 1 R is an amino acid sequence comprising from 1 to about 40 amino acids, each amino acid being independently selected from the group consisting of naturally occurring amino acids and amino acid analogs. 2is an amino acid sequence comprising from 1 to about 40 amino acids, where each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs, and x and y are independently selected and equal to 0 or 1.

[0042] According to some embodiments of the invention, the polypeptide comprises at least one D-amino acid.

[0043] According to some embodiments of the invention, the polypeptide is less than 50 amino acids in length.

[0044] According to some embodiments of the invention, the polypeptide is less than 20 amino acids in length.

[0045] According to some embodiments of the invention, the polypeptide is conjugated to a permeabilizing moiety or a stabilizing moiety.

[0046] According to some embodiments of the invention, the polypeptide and the SIRT1 activator are provided in a co-formulation.

[0047] According to some embodiments of the invention, the polypeptide and the agent are provided in a co-formulation.

[0048] According to some embodiments of the invention, the polypeptide and bumetanide are provided in a co-formulation.

[0049] According to some embodiments of the invention, the polypeptide and the cannabinoid are provided in a co-formulation.

[0050] According to some embodiments of the invention, the polypeptide and ketamine are provided in a co-formulation.

[0051] According to some embodiments of the invention, the polypeptide and the SIRT1 activator are provided in separate formulations.

[0052] According to some embodiments of the invention, the polypeptide and the agent are provided in separate formulations.

[0053] According to some embodiments of the invention, the polypeptide and bumetanide are provided in separate formulations.

[0054] According to some embodiments of the invention, the polypeptide and the cannabinoid are provided in separate formulations.

[0055] According to some embodiments of the invention, the polypeptide and ketamine are provided in separate formulations.According to some embodiments of the invention, the subject is female.

[0056] According to some embodiments of the invention, the subject is male.

[0057] According to some embodiments of the invention, the subject is under 18 years of age.

[0058] According to some embodiments of the invention, the subject is over 60 years of age.

[0059] According to some embodiments of the invention, the disease is associated with aging.

[0060] According to some embodiments of the invention, the disease is an inflammatory disease.

[0061] According to some embodiments of the invention, the disease is a neurodegenerative disease or a cognitive disorder.

[0062] According to some embodiments of the invention, the disease is Alzheimer's disease.

[0063] According to some embodiments of the invention, the disease is autism spectrum disorder and / or intellectual disability.

[0064] According to some embodiments of the invention, the disease is ADNP syndrome.

[0065] According to some embodiments of the invention, the disease is selected from the group consisting of stress, anxiety, bipolar disorder, schizophrenia and aggression.

[0066] According to some embodiments of the invention, the disease is selected from the group consisting of hypertension, swelling, congestive heart failure, liver disease and kidney disease.

[0067] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of the invention, representative methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. Furthermore, the materials, methods, and examples are merely illustrative and are not intended to be necessarily limiting. [Brief description of the drawings]

[0068] Some embodiments of the present invention are herein described, by way of example only, with reference to the accompanying drawings. With particular reference now to the drawings in detail, it is stressed that the details shown are by way of example and are for the purpose of an illustrative discussion of embodiments of the invention. In this regard, the description taken in conjunction with the drawings will make apparent to those skilled in the art how embodiments of the present invention may be practiced.

[0069] 1A to 1G show that ADNP interacts with SIRT1. [Figure 1A] Schematic diagram of functional protein regions depicted along with full length human ADNP and SIRT1 amino acid coding sequences. ADNP sequence motifs are extended from this description. 15 The SSIP (SEQ ID NO:51) motif that binds EB1 / EB3 is labeled on the SIRT1 scheme. [Figure 1B]The best docking poses of SIRT1 (pink ribbon) and EB1 homodimer (white ribbon) are shown. The binding motif in SIRT1 is shown as cyan spheres. Residues that bind to the SXIP (SEQ ID NO:52) motif in EB1 are shown as white spheres (219 in monomer A and 220, 223 in monomer B). [Figure 1C] Images of HEK293T cells immunostained with ADNP (red) and SIRT1 (green) specific antibodies are shown. Cell nuclei are visualized with DAPI (blue). Nuclear colocalization of the two proteins is shown in yellow. Quantitative analysis of ADNP-SIRT1 combined staining colocalization is shown in the graph with a x63 oil immersion lens. [Figure 1D] Images of iPSC37 derived from a healthy individual differentiated into neural progenitor cells in suspension and stained as in Figure 1C are shown. [Figure 1E] Figure 1 shows the endogenous Co-IP assay of ADNP and SIRT1 in differentiated SH-SY5Y cells. EB1 or EB3 antibodies were conjugated to affinity beads, and the successive elution fractions (flow-through=FT, wash=W1, W2, W3, elution=E) were further analyzed by immunoblotting (IB) with ADNP, SIRT1, EB1 and EB3 antibodies (IB:ADNP, IB:SIRT1, IB:EB1, IB:EB3). In addition, a column with free agarose beads was used as a negative control (IP:IgG). [Figure 1F]Co-IP assay of neural progenitor cell extracts subjected to EB1 antibody column. Sequential antibody reactions (16 h, 40° C.) were performed on the same blot (after striping). Blots indicated with MW markers (kDa) did not detect SIRT1 in the elution (expected band size: 81 kDa, or observed band size: 110 kDa www(dot)abcam(dot)com / sirt1-antibody-19a7ab4-ab110304(dot)html), lower exposure (2 min, Fusion Fx, Collegien, France) upper panel, higher exposure (5 min), lower panel. Further exposure of the blot to EB1 antibody (1 h, Fugi Medical X-ray film, Japan) showed EB1 in the FT and elution (E), suggesting column saturation. Potentially non-specific higher molecular weight bands are observed, probably due to previous antibody exposure of the blot, despite extensive washing. [Figure 1G] The effect of NAP (SEQ ID NO:2) is shown. To enhance EB1 binding to partner proteins, 3 mg of NAP was added to FT and the mixture was incubated with EB1 agarose-conjugated antibody. After blotting, the SIRT1 predicted size band was seen in the eluted material (E1, EB1-bound), albeit at a low detection level (film exposure times were similar to those in Fig. 1F). The lower panel shows exposure of the blot film to tau antibody (two incubation times, 1 h, 250°C, and 16 h, 40°C). Figures 2A-C show RNA-seq identification of ADNP-SIRT1 colocalization at the human single-cell level. [Figure 2A] FIG. 1 shows single-cell RNA sequencing data from a human cortical specimen (PRJNA29546945) visualized using the UCSC Cell Browser (www.cells.ucsc.edu / ?ds=cortex-dev.) ADNP and SIRT1 expressing cells are labeled with black circles. [Figure 2B-1]ADNP and SIRT1 total bulk RNA expression levels from 48 human tissues (GTEx) data visualized with t-SNE using UCSC Cell Browser (www(dot)cells(dot)ucsc(dot)edu / ?ds=gtex8). [Figure 2B-2] ADNP and SIRT1 total bulk RNA expression levels from 48 human tissues (GTEx) data visualized with t-SNE using UCSC Cell Browser (www(dot)cells(dot)ucsc(dot)edu / ?ds=gtex8). [Figure 2C] Single-cell RNA sequencing data (GSE3655246) from 124 cells of human embryonic stem cells and human preimplantation embryos are shown. Single-cell expression levels of ADNP, SIRT1, MAPR1 and MAPR3 were analyzed using the Single Cell Expression Atlas47. Figures 3A-C demonstrate that ADNP and SIRT1 are co-regulated at the transcriptional level and both control specific histone H3 modifications. [Figure 3A] Histone H3 modification screening from the hippocampus of 5-month-old female Adnp+ / - and Adnp+ / + mice is shown (red box H3K79me2). [Figure 3B-1]WashU Epigenome Browser diagram of ADNP, SIRT1, and MAPRE1 showing chip-seq (Chromatin Immuno Precipitation Sequencing) binding peaks of H3K79me2 (GSM733653), ADNP (GSE105573), HDAC2 (GSM1003447), YY1 (GSM803470, GSM803446), and SMARCA4 (GSE91946) in the erythroblast cell line K562. Boxes indicate histone modifications adjacent to the ADNP peak. Histone peaks are separated for peaks proximal (<1 kb) to the Transcription Start Site (TSS) and distal to the TSS, each 2 kb around the position with most sequence reads and Factorbook50 analyzed. [Figure 3B-2] WashU Epigenome Browser diagram of ADNP, SIRT1, and MAPRE1 showing chip-seq (Chromatin Immuno Precipitation Sequencing) binding peaks of H3K79me2 (GSM733653), ADNP (GSE105573), HDAC2 (GSM1003447), YY1 (GSM803470, GSM803446), and SMARCA4 (GSE91946) in the erythroblast cell line K562. Boxes indicate histone modifications adjacent to the ADNP peak. Histone peaks are separated for peaks proximal (<1 kb) to the Transcription Start Site (TSS) and distal to the TSS, each 2 kb around the position with most sequence reads and Factorbook50 analyzed. [Figure 3B-3]WashU Epigenome Browser diagram of ADNP, SIRT1, and MAPRE1 showing chip-seq (Chromatin Immuno Precipitation Sequencing) binding peaks of H3K79me2 (GSM733653), ADNP (GSE105573), HDAC2 (GSM1003447), YY1 (GSM803470, GSM803446), and SMARCA4 (GSE91946) in the erythroblast cell line K562. Boxes indicate histone modifications adjacent to the ADNP peak. Histone peaks are separated for peaks proximal (<1 kb) to the Transcription Start Site (TSS) and distal to the TSS, each 2 kb around the position with most sequence reads and Factorbook50 analyzed. [Figure 3C] Motif-enriched sequences of ADNP, HDAC2, YY1 and SMARCA4 are shown.Figures 4A-4C show that ADNP and SIRT1 correlate and interact with histone remodeling complex proteins. [Figure 4A] STRING protein-protein interaction network analysis of histone interacting protein network with SIRT1 and ADNP (p-value < 1.0e-16, from STRING database, www(dott)string-db(dot)org / ). [Figure 4B] Pearson correlation of SIRT1 and ADNP expression levels (Reads Per Kilobase of exon per Million mapped reads, RPKM) in human tissues from 27 normal tissue samples from 95 human individual public dataset PRJEB433753. Inset shows Pearson correlation of Sirt1 and Adnp expression levels from Adnp+ / - and Adnp+ / + mouse hippocampus (GSE72664)12 (N=23). [Figure 4C]Correlation matrix plot of RNA expression levels of key histone-interacting proteins from postmortem brain structures across human brain development. Data are from BrainSpan atlas51. Positive correlations are displayed in blue scale and negative correlations are displayed in red scale at a significance level of p<0.05. Numbers represent correlation coefficient values. Open boxes represent non-significant correlations. [Diagram 5] AD postmortem brains show significant dysregulation in the ADNP / SIRT1 network. Correlation matrix plot of postmortem brain expression levels of key histone interacting proteins from Alzheimer's disease patients or controls (GSE5281). Data were obtained from six postmortem brain regions: entorhinal cortex, hippocampus, medial temporal gyrus, posterior cingulate gyrus, superior frontal gyrus, and primary visual cortex (including 74 samples from controls and 87 samples from AD patients). Positive correlations are displayed in blue scale and negative correlations in red scale. Asterisks represent significance levels ***p<0.001, **p<0.01, *p<0.05. [Figure 6] FIG. 1 shows the ADNP-SIRT1 complex network generated using the software BioRender(dot)com.

[0070] Detailed Description of Certain Embodiments of the Invention The present invention, in some aspects, relates to compositions and articles comprising Activity Dependent Neurotrophic Factor (ADNF) polypeptides.

[0071] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by way of examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0072] While investigating the mechanism of action of the ADNP gene product, the inventors have uncovered extensive interactions between activity-dependent neurotrophic factor (ADNF) and mammalian nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase SIRT1 (Sirtuin 1). The physical interaction between the two polypeptides ADNP and SIRT1 was found to be mediated through the mutual binding of microtubule end-binding protein 1 (EB1) (see FIG. 1B and Example 1 below). Further investigations revealed nuclear co-localization of ADNP and SIRT1, shared histone-modifying activity and coordinated tissue-specific expression, as well as coordinated expression patterns for a number of proteins associated with both ADNP and SIRT1 (see Examples 2 and 3 below), pointing to their involvement in various signaling pathways. Now, component factors of these interactions can be used to increase the activity of ADNF polypeptides and find previously undisclosed clinical applications.

[0073] Given the importance of ADNP to functions related to autism spectrum disorder and intellectual disability diseases, the newly recognized physical interactions, co-localization, mutual biochemical functions, and coordinate expression and protein interactions between ADNP and SIRT1 suggest a role for SIRT1 and similar molecules in autism spectrum disorder, intellectual disability, and certain neurodegenerative diseases.

[0074] Thus, in some embodiments of the invention, the present invention is directed to a variety of disorders, including but not limited to, Activation-Dependent Neuroprotective Protein (ADNP) syndrome (also known as Helsmoortel-Van der Aa syndrome), Dravet syndrome, Fragile X syndrome, Snaptic Ras GTPase activating protein 1 (SYNGAP1)-associated intellectual disability, Phelan-McDermid syndrome, Glutmate Ionotropic Receptor NMDA (GRIN) disorders, Chromodomain Helicase Binding Protein (CHD8)-associated disorders, Dual Specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A) syndrome, Pogo Transposable Element with ZNF Domain (POGZ) syndrome, Forkhead Box P1 (FOXP1) syndrome, Solute Carrier Family 5 syndrome, A method of treating a disease in a subject in need of treatment is provided, the method comprising administering to the subject a therapeutically effective amount of a SIRT1 activator, thereby treating the disease in the subject. The method comprises administering to the subject a therapeutically effective amount of a SIRT1 activator, the method comprising administering to the subject a therapeutically effective amount of a SIRT1 activator, thereby treating the disease in the subject.

[0075] As used herein, the term "SIRT1" (also known as Sirtuin 1 and NAD-dependent deacetylase Sirtuin-1) refers to the expression product, e.g., RNA or protein, of the SIRT1 gene (gene ID 23411). The SIRT1 protein converts protein-derived acetyl groups to NAD + to the ADP-ribose moiety to form O-acetyl-ADP-ribose, EC No. 2.3.1. Methods for determining the catalytic activity of SIRT1 are well known in the art and include, for example, 3 These include isotopic assays such as a charcoal binding assay, which measures acetate release from OAADPr hydrolyzed using a 3H-labeled acetyl-lysine substrate. 14 [C]NAM release assay and Fluor de Lys assay™ (BIOMOL / Enzo), which measures the deacetylation of an acetyl-lysine peptide conjugated to aminomethylcoumarin (AMC). Kits for assaying SIRT1 activity are also commercially available, e.g., from BIOMOL / Enzo, Sigma, Biovision, and Abcam.

[0076] According to certain embodiments, the SIRT1 is human SIRT1 as provided in the following catalog numbers NM_001142498, NM_001314049, NM_012238, NP_001135970, NP_001300978, NP_036370.

[0077] Many SIRT1 activators are known to those skilled in the art. For example, SIRT1 activators are described in U.S. Patent Publication Nos. 20130085155, 20120197013, 20120165330, 20120108585, 20120022254, 20110306612, 20110306609, 20110263564, 2011025717, and the like. No. 4, No. 20110152254, No. 20110130387, No. 20110077248, No. 20110039847, No. 20110015192 , No. 20110009496, No. 20100215632, No. 20090163476, No. 20090105246, No. 20090099170, No. 20090069301, 20090012080, 20080249103, 20070043050, 20070037865, 20 070037827, 20070037809, 8,343,997, 8,268,862, 8,247,565, 8,178,536, Nos. 8,163,908, 8,093,401, 8,088,928, 8,044,198, 7,998,974, 7,893,086, 7,855,289, 7,829,556, and 7,345,178, each of which is incorporated herein by reference in its entirety.SIRT1 activators are further described in Dai et al., J Biol Chem, 285(43):32695-32703,2010, which is incorporated herein by reference in its entirety. Additional SIRT1 activators are provided as formulas I-XXXVIII in U.S. Pat. No. 8,044,198 (incorporated herein by reference), U.S. Pat. No. 20150133527 (London et al., Schiedel et al., Med Res Rev 0:1-54, 2017) (incorporated herein by reference).

[0078] Non-limiting examples of SIRT1 activators that may be used with some embodiments of the present invention include resveratrol, quercetin, butein, beverine, curcumin, fisetin, honokiol, YK3-237, SRT1720, SRT1460, SRT2183, STAC-5, STAC-9, STAC-10, BML-278, SRT2104 (GSK2245840), SIRT1 activator 3, STAC8, MC2563, SCIC2, SCIC2.1, oxazolo(4,5-b)pyridine, pyrrolo(3-2b)quinoxaline, benzimidazole, pyridoxazole, and / or piceatannol.

[0079] Thus, in some embodiments, the SIRT1 activator is selected from the group consisting of resveratrol, quercetin, butein, beverine, curcumin, fisetin, honokiol, YK3-237, SRT1720, SRT1460, SRT2183, STAC-5, STAC-9, STAC-10, BML-278 and piceatannol.

[0080] In some embodiments, the SIRT1 activator is a small molecule.

[0081] As used herein, the term "small molecule" refers to organic compounds of low molecular weight (<900 Daltons) having a size on the order of 1 nm that can regulate biological processes.

[0082] In some embodiments, the SIRT1 activator is selected from the group consisting of resveratrol, quercetin, butein, beverin, curcumin, fisetin, honokiol, YK3-237, SRT1720, SRT1460, SRT2183, STAC-5, STAC-9, STAC-10, BML-278, and piceatannol, or an analog or derivative thereof. In certain embodiments, the SIRT1 activator is resveratrol, or an analog or derivative thereof.

[0083] In yet other embodiments, the SIRT1 activator is NAD+ or an analog or derivative thereof.

[0084] As used herein, "NAD+" refers to the oxidized form of nicotinamide adenine dinucleotide (NAD). NAD is a central coenzyme in metabolism consisting of nicotinamide and adenine linked through their phosphate groups.

[0085] According to some embodiments of the invention, the SIRT1 activator is nicotinamide riboside (NR), the biosynthetic precursor of NAD.

[0086] Also contemplated in the present invention is an article of manufacture comprising as active ingredients an ADNF polypeptide having neurotrophic / neuroprotective activity in an in vitro cortical neuronal culture assay and a SIRT1 activator. In some embodiments, the ADNF polypeptide and the SIRT1 activator are provided in a co-formulation in the article of manufacture. In other embodiments, the ADNF polypeptide and the SIRT1 activator are provided in separate formulations.

[0087] The present inventors have elucidated the role of ADNF polypeptides and SIRT1 in chromatin remodeling, particularly in histone modifications, such as histone methylation. Many of the neurodevelopmental conditions associated with ADNP share components of chromatin remodeling with the aging process, and therefore anti-aging agents may be useful in therapy.

[0088] Thus, in a further embodiment, there is provided a method of treating a disorder selected from the group consisting of ADNP syndrome, Dravet syndrome, Fragile X syndrome, SYNGAP1 associated intellectual disability, Phelan-McDermid syndrome, GRIN disorder, CHD8 associated disorder, DYRK1A syndrome, POGZ syndrome, FOXP1 syndrome, SLC5A1 associated disorder, Coffin-Siris syndrome, ARID1B associated syndrome, KMT5B syndrome, PTEN autism syndrome, Rett syndrome, Okihiro syndrome plus developmental delay, Angelman syndrome, Noonan syndrome, Kleefstra syndrome, and Smith-Maginnis syndrome in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of an anti-aging agent, thereby treating the disorder in the subject.

[0089] Non-limiting examples of anti-aging agents include vitamins (e.g., A, C, and B3), polyphenols, flavonoids, cell regulators such as retinol, antioxidants such as peptides and growth factors, etc. In some embodiments, the anti-aging agent is selected from the group consisting of rapamycin, metformin, melatonin, carnosine, nicotinamide mononucleotide, δ-sleep inducing peptides, and low molecular weight klotho enhancers, or analogs or derivatives thereof.

[0090] Similarly, an article of manufacture is contemplated in the present invention that includes as active ingredients an ADNF polypeptide having neurotrophic / neuroprotective activity in an in vitro cortical neuronal culture assay and an anti-aging agent, where the anti-aging agent is not an antioxidant. In some embodiments, the ADNF polypeptide and the anti-aging agent are provided in a co-formulation in the article of manufacture. In other embodiments, the ADNF polypeptide and the anti-aging agent are provided in separate formulations.

[0091] Also contemplated are methods or articles of manufacture in which the anti-aging agent comprises behavioral modification such as a calorie restricted diet, smoking cessation, exercise and / or improved sleep habits.

[0092] In some embodiments, the anti-aging agent is a SIRT1 activator.

[0093] The present inventors have identified immune modulators among factors (e.g., HDAC2, SMARCA4, and YY1) whose expression correlates with ADNP and SIRT1. Thus, in some aspects of some embodiments, a method of treatment in a subject in need of treatment for a disease selected from the group consisting of ADNP syndrome, Dravet syndrome, fragile X syndrome, SYNGAP1-associated intellectual disability, Phelan-McDermid syndrome, GRIN disorder, CHD8-associated disorder, DYRK1A syndrome, POGZ syndrome, FOXP1 syndrome, SLC5A1-associated disorder, Coffin-Siris syndrome, ARID1B-associated syndrome, KMT5B syndrome, PTEN autism syndrome, Rett syndrome, Okihiro syndrome + developmental delay, Angelman syndrome, Noonan syndrome, Kleefstra syndrome, and Smith-McGinnis syndrome, comprising administering to the subject a therapeutically effective amount of an immune modulator selected from the group consisting of a chemokine receptor modulator, an immune checkpoint modulator, and a cytokine, thereby treating the disease in the subject, is provided. In some embodiments, the cytokine is not IFNβ.

[0094] As used herein, the term "chemokine receptor modulator" refers to any agent that alters the function of a chemokine receptor, including but not limited to receptors for the chemokines CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, XCR1, ACKR1, ACKR2, ACKR3, ACKR4, and CCRL2.

[0095] In some embodiments, the chemokine receptor is selected from the group consisting of CCR5 and CXCR4.

[0096] In some embodiments, the modulator is an inhibitor. In other embodiments, the chemokine receptor modulator is selected from the group consisting of maraviroc, leronlimab, aplaviroc, vicriviroc, plerixafor, mavorixafor, BL-8040, and TGO-0054, or analogs or derivatives thereof. In certain embodiments, the chemokine receptor modulator is selected from the group consisting of maraviroc and plerixafor, or analogs or derivatives thereof.

[0097] Also contemplated in the present invention is an article of manufacture comprising as active ingredients an ADNF polypeptide having neurotrophic / neuroprotective activity in an in vitro cortical neuronal culture assay and an immunomodulatory agent selected from the group consisting of a chemokine receptor modulator, an immune checkpoint modulator, and a cytokine, wherein the cytokine is not IFNβ. In some embodiments, the ADNF polypeptide and the immunomodulatory agent are provided in a co-formulation in the article of manufacture. In other embodiments, the ADNF polypeptide and the immunomodulatory agent are provided in separate formulations.

[0098] As used herein, the term "immunomodulatory agent" includes interferons, antigens, tumor phagocytosis inducers, and other immune enhancing agents (e.g., immune checkpoint inhibitors).

[0099] Interferons include, but are not limited to, interferon alpha, interferon alpha-2a, interferon alpha-2b, interferon beta, interferon gamma-1a, ACTIMMUNE® (interferon gamma-1b), or interferon gamma-n1, combinations thereof, and the like.

[0100] Tumor phagocytosis inducers include, but are not limited to, anti-CD47 monoclonal antibodies (e.g., Hu5F9-G4, CC-90002, ZF1, AMMS4-G4, IBI188, SRF231), anti-SIRPα fusion proteins (e.g., TTI-621, TTI-622), anti-SIRPα monoclonal antibodies (e.g., OSE-172), anti-CD47 / anti-tumor associated antigen bispecific antibodies, and inhibitors of leukocyte immunoglobulin-like receptor B1 (LILRB1) that binds to major histocompatibility complex class 1 β2-microglobulin (MHC class 1 β2M).

[0101] Anti-CD47 / anti-tumor associated antigen bispecific antibodies include, but are not limited to, anti-CD47 / CD19 bispecific antibodies (e.g., TG-1801), anti-CD47 / mesothelin bispecific antibodies (e.g., NI-1801), anti-CD47 / 4-1BB bispecific antibodies (e.g., DSP107), anti-CD47 / CD20 bispecific antibodies, anti-CD47 / CD33 bispecific antibodies (e.g., HMBD004).

[0102] Immune checkpoint inhibitors include, but are not limited to, PD-1 inhibitors (e.g., nivolumab, pidilizumab, sintilimab), PD-L1 inhibitors (e.g., atezolizumab, avelumab, durvalumab, BMS-936559), CTLA4 inhibitors (e.g., ipilimumab, tremelimumab) or IDO inhibitors (e.g., indoximod, epacadostat).

[0103] Other immunomodulatory agents include ALFAFERONE®, BAM-002, BEROMUN® (tasonermin), BEXXAR® (tositumomab), CamPath® (alemtuzumab), CTLA4 (cytotoxic lymphocyte antigen 4), decarbazine, denileukin, epratuzumab, GRANOCYTE® (lenograstim), lentinan, leukocyte alpha interferon, imiquimod, MDX-010, melanomavaccine, mitumomab, molgramostim, MYLOTARG™.RTM™® (gemtuzumab ozogamicin), NEUPOGEN® (filgrastim), OncoV These include AC-CL, OvaRex® (oregovomab), pertuzumab (Y-muHMFG), PROVENGE®, sargramostim, sizofiran, teceleukin, TheraCys®, ubenimex, Virulizin, Z-100, WF-10, PROLEUKIN® (aldesleukin), ZADAXIN® (thymalfasin), ZENAPAX® (daclizumab), ZEVALIN® (90Y-ibritumomab tiuxetan), and the like, including, but not limited to, STING (stimulator of interferon genes) and NOD (nucleotide-binding oligomerization domain-like receptor) agonists.

[0104] As used herein, the term "cytokine" refers to a superfamily of proteins that are essential in signaling networks between cells and that regulate the immune system. Cytokines include, but are not limited to, interleukins (e.g., IL-1, IL-2, IL-18, IL-4, IL-7, GCSF, etc.), interferons (e.g., IFN-α, IFN-β, IFN-γ, TNF, CD154, etc.), TGF, and hematopoietins such as Epo, Tpo, and SCF.

[0105] Also contemplated herein is an article of manufacture or method of the invention, wherein the cytokine is selected from the group consisting of IL-6, IL-10 and TNFα.

[0106] According to one aspect of some embodiments of the present invention there is provided an article of manufacture comprising as active ingredients an ADNF polypeptide having neurotrophic / neuroprotective activity in an in vitro cortical neuronal culture assay and bumetanide or an analog or derivative thereof.

[0107] Bumetanide (also known as Bumdex®) is a drug used to treat hypertension and edema, edema resulting from heart failure, liver failure and / or kidney disease. Some representative bumetanide analogs are described in U.S. Patent No. 9,682,928 to Partridge et al.

[0108] In some embodiments, the ADNF polypeptide and bumetanide or an analog or derivative thereof are provided in a co-formulation in an article of manufacture, hi other embodiments, the ADNF polypeptide and bumetanide or an analog or derivative thereof are provided in separate formulations.

[0109] According to one aspect of some embodiments of the present invention, there is provided an article of manufacture comprising as active ingredients an ADNF polypeptide having neurotrophic / neuroprotective activity in an in vitro cortical neuronal culture assay and a cannabinoid.

[0110] As used herein, the term "cannabinoid" refers to a diverse class of chemical compounds that act on cannabinoid receptors on cells that inhibit neurotransmitter release in the brain. Ligands for these receptor proteins include endocannabinoids (produced naturally in the body by humans and animals), phytocannabinoids (found in cannabis and other plants), and synthetic cannabinoids (manufactured artificially). There are at least 85 different cannabinoids isolated from cannabis that exhibit a variety of effects (El-Alfy et al., Pharmacology Biochemistry and Behavior, Vol. 95(4), 2010, pp. 434-442). Exemplary cannabinoids include, but are not limited to, tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), cannabicyclol (CBL), cannabivarin (CBV), tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), cannabichromevarin (CBCV), cannabigerovarin (CBGV), and cannabigerol monomethyl ether (CBGM). In certain embodiments, the cannabinol is tetrahydrocannabinol (THC) or cannabidiol (CBD).

[0111] In some embodiments, the ADNF polypeptide and the cannabinoid are provided in a co-formulation in the article of manufacture, hi other embodiments, the ADNF polypeptide and the cannabinoid are provided in separate formulations.

[0112] According to one aspect of some embodiments of the present invention, there is provided an article of manufacture comprising as active ingredients an ADNF polypeptide having neurotrophic / neuroprotective activity in an in vitro cortical neuronal culture assay and ketamine or an analog or derivative thereof.

[0113] As used herein, the terms "ketamine," "ketamine analog," or "ketamine derivative" refer to ketamine, norketamine, 6-hydroxynorketamine, or pharma- ceutically acceptable salts thereof.

[0114] As used herein, the term "ketamine" includes ketamine in its racemic (R / S) form, its R-(-) enantiomerically pure form, or its S-(+) enantiomerically pure form. As used herein, the term "norketamine" includes norketamine in its racemic (R / S) form, its R-(-) enantiomerically pure form, or its S-(+) enantiomerically pure form. As used herein, "enantiomerically pure" refers to a composition that consists substantially of a single isomer (i.e., substantially free of the opposite isomer), preferably 90%, 92%, 95%, 98%, 99%, or 100% (w / w) of a single isomer. For example, if an article of manufacture contains enantiomerically pure R-(-)-ketamine, the article of manufacture may contain at least 95% (w / w) S-(+)-ketamine and less than 5% (w / w) R-(-)-ketamine.

[0115] In some aspects, the ADNF polypeptide and ketamine or an analog or derivative thereof are provided in a co-formulation in the article of manufacture, hi other embodiments, the ADNF polypeptide and ketamine or an analog or derivative thereof are provided in separate formulations.

[0116] Also contemplated is a method of treating a disease in a subject in need of treatment that can benefit from treatment with an active ingredient of an article of manufacture of the invention, comprising administering to the subject a therapeutically effective amount of a component active ingredient of the article of manufacture described herein, thereby treating the disease in the subject.

[0117] In certain embodiments, the disease is an aging-related disease.

[0118] In other embodiments, the disease is an inflammatory disease.

[0119] In yet other embodiments, the disease is a neurodegenerative disease or a cognitive disorder.

[0120] In some embodiments, the disease is Alzheimer's disease.

[0121] In other embodiments, the disease is an autism spectrum disorder and / or intellectual disability.

[0122] In some embodiments, the disease is ADNP syndrome.

[0123] In some embodiments, the disease is selected from the group consisting of stress, anxiety, bipolar disorder, schizophrenia, and aggression.

[0124] In other embodiments, the disease is selected from the group consisting of hypertension, swelling, congestive heart failure, liver disease, and kidney disease.

[0125] As used herein, the term "treating" refers to inhibiting, substantially arresting, slowing, or reversing the progression of a medical condition (e.g., autism spectrum disorder, intellectual disability, Alzheimer's disease, ADNP syndrome, Dravet syndrome, Fragile X syndrome, SYNGAP1 associated intellectual disability, Phelan-McDermid syndrome, GRIN disorder, CHD8 associated disorder, DYRK1A syndrome, POGZ syndrome, FOXP1 syndrome, SLC5A1 associated disorder, Coffin-Siris syndrome, ARID1B associated syndrome, KMT5B syndrome, PTEN autism syndrome, Rett syndrome, Okihiro syndrome + developmental delay, Angelman syndrome, Noonan syndrome, Kleefstra syndrome, and Smith-Magenis syndrome, stress, anxiety, schizophrenia, high blood pressure, liver disease, and kidney disease, and other diseases, disorders, or conditions that may benefit from treatment with the active ingredients of the articles of manufacture of the invention), substantially ameliorating the symptoms of the medical condition, and / or improving survival in a subject diagnosed with the medical condition. Those of skill in the art will appreciate that a variety of methodologies and assays may be used to assess the onset of a condition or the reduction or regression of a condition, as further disclosed herein.

[0126] As used herein, the term "prevent" refers to keeping a disease state from occurring in a subject who has not yet been diagnosed with the disease state and / or preventing the onset of symptoms associated with a disease state before the disease state occurs.

[0127] As used herein, the term "improvement" or "improving" refers to reducing or alleviating the severity, frequency, or duration of a negative aspect of a disease, condition, or disorder in a subject, or increasing or causing (the frequency, intensity, or duration of) a positive, beneficial, or desirable aspect of the subject's health and / or well-being associated with the disease, condition, or disorder. Some non-limiting examples of improvement following treatment with a SIRT1 activator, anti-aging agent, immunomodulator, ADNF polypeptide, or article of manufacture according to the present invention are improved intelligence and / or cognitive ability, reduced autism spectrum behavior, reduced blood pressure.

[0128] Non-limiting examples of diseases that may be treated according to some embodiments of the present invention include inflammatory diseases, neurodegenerative diseases, cognitive disorders, autism spectrum disorders, psychiatric disorders, cytoskeletal disorders (e.g., Dravet Syndrome, Rett Syndrome and Fragile X Syndrome), hypertension and swelling (e.g., as a result of heart failure, liver failure or kidney disorders such as nephrotic syndrome), autoimmune diseases, allergic diseases, infectious diseases, transplant rejection diseases and cancerous diseases.

[0129] As used herein, the term "cognitive disorder" encompasses both intellectual disability and cognitive impairment (typically associated with a psychiatric disorder or neurodegenerative disease).

[0130] As used herein, the term "Intellectual Disability (ID)", also known as General Learning Disability or Mental Retardation (MR), refers to a generalized neurodevelopmental disorder characterized by significantly impaired intellectual and adaptive functioning.

[0131] Non-limiting examples of neurodegenerative diseases or cognitive disorders include diseases of the central motor system, including degenerative conditions affecting the basal ganglia (Huntington's disease, Wilson's disease, striatonigral degeneration, corticobasal ganglionic degeneration), Tourette's syndrome, Parkinson's disease, progressive supranuclear palsy, progressive bulbar palsy, familial tonic paraplegia, spinal muscular atrophy, ALS and its variants, dentatorubral atrophy, olivopontocerebellar atrophy, paraneoplastic cerebellar degeneration, as well as dopamine poisoning, sensory neuronal degeneration, and the like. diseases affecting the brain, such as Friedreich's ataxia, diabetes, peripheral neuropathy, retinal neurodegeneration, diseases of the limbic and cortical systems, such as cerebral amyloidosis, Pick's atrophy, Rett's syndrome; neurodegenerative conditions involving multiple neuronal systems and / or the brain stem, such as Alzheimer's disease, Parkinson's disease, AIDS-related dementia, Leigh's disease, diffuse Lewy body disease, multiple sclerosis, epilepsy, multiple system atrophy, Guillain-Barré syndrome, lipofuscinosis, and other lysosomal diseases; neurodegenerative conditions including cerebrospinal fluid storage disorders, late degenerative stages of Down's syndrome, Alpers' disease, vertigo as a result of CNS degeneration, ALS, corticobasal degeneration, and progressive supranuclear palsy, developmental delay and learning disabilities, Down's syndrome, Fragile X syndrome, Klinefelter's syndrome, Prader-Willi syndrome, cri-a-cat syndrome, and conditions associated with oxidative stress-induced neuronal cell death, such as (i) alcoholism, neurodegenerative disorders in the locus coeruleus, cerebellum, and cholinergic basal forebrain (ii) degeneration of cerebellar and cortical neurons leading to aging, cognitive and motor disorders, and (iii) conditions associated with chronic alcohol or drug abuse, including chronic amphetamine abuse, degeneration of basal ganglia neurons leading to movement disorders; pathological changes resulting from localized trauma such as stroke, focal ischemia, vascular insufficiency, hypoxic-ischemic encephalopathy, hyperglycemia, hypoglycemia, closed head injury, and direct trauma; Post Traumatic Stress Disorder (PTSD); conditions occurring as a negative side effect of therapeutic agents and treatments (e.g., degeneration of cingulate cortex and entorhinal cortex neurons in response to anticonvulsant doses of antagonists of the NMDA class of glutamate receptors).

[0132] Non-limiting examples of autism spectrum disorders and / or intellectual disabilities include ADNP syndrome, Dravet syndrome, Fragile X syndrome, Down syndrome, SYNGAP1 syndrome, POGZ syndrome (White-Sutton syndrome), CHD8 syndrome, SCN2A syndrome, ARID1B syndrome, Phelan-McDermid syndrome, NRXN1 syndrome, DYRK1A syndrome, GRIN disorder, POGZ (White-Sutton syndrome), FOXP1 syndrome, SLC5A1-associated disorder, Coffin-Siris syndrome, ARID1B-associated syndrome, KMT5B syndrome, PTEN autism syndrome, CHD2 syndrome, Rett syndrome, Okihiro syndrome + developmental delay, Angelman syndrome, Noonan syndrome, Kleefstra syndrome, and Smith-Magenis syndrome.

[0133] According to certain embodiments, the disease is not an autism spectrum disorder.

[0134] According to a particular embodiment, the disease is not ADNP syndrome.

[0135] Non-limiting examples of mental disorders include mood disorders (e.g., major depressive disorder (i.e., unipolar disorder), mania, dysphoria, bipolar disorder, dysthymia, cyclothymia), psychotic disorders (e.g., schizophrenia, schizoaffective disorder, schizophreniform disorder, delusional disorder, brief psychotic disorder, and shared psychotic disorder), personality disorders, post-traumatic stress disorder (PTSD), aggression, anxiety disorders (e.g., obsessive-compulsive disorder and attention deficit disorder), and other mental disorders such as substance-related disorders, childhood disorders, dementia, adjustment disorder, delirium, vascular dementia, Tourette's disorder, etc., as described in the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV) (see also review, Benitez-King G et al., Curr Drug Targets CNS Neurol Disord. December 2004, 3(6):515-33). Typically, such disorders have complex genetic or biochemical components.

[0136] Inflammatory diseases - including, but not limited to, chronic inflammatory diseases and acute inflammatory diseases.

[0137] Hypersensitivity-related inflammatory disorders Examples of hypersensitivity include, but are not limited to, type I hypersensitivity, type II hypersensitivity, type III hypersensitivity, type IV hypersensitivity, immediate hypersensitivity, antibody-mediated hypersensitivity, immune complex-mediated hypersensitivity, T-lymphocyte-mediated hypersensitivity, and DTH.

[0138] Type I or immediate hypersensitivity, such as asthma.

[0139] Type II allergy includes inflammatory diseases, autoimmune inflammatory diseases, joint inflammatory diseases (Krenn V., Histol Histopathol, 2000, July, 15(3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel, Arthritis Res 2001, 3(3):189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J., Immunol Res 1998, 17(1-2):49), sclerosis, systemic sclerosis (Renaudineau Y., Clin Diagn Lab Immunol. 1999, March, 6(2):156), Chan OT., Immunol Rev. 2001, 3(3):189), and inflammatory diseases. 1999 July, 169:107), gland diseases, gland autoimmune diseases, pancreatic autoimmune diseases, diabetes, type I diabetes (Zimmet P. Diabetes Res Clin Pract 1996 October, 34 Suppl:S125), thyroid diseases, autoimmune thyroid diseases, Graves' disease (hyperthyroidism) (Orgiazzi J. Endocrinol Metab Clin North Am 2000 June 2, 9(2):339), thyroiditis, autoimmune autoimmune thyroiditis (Braley-Mullen H. et al. J Immunol 2000 December 15, 165(12):7262), Hashimoto's thyroiditis (Toyoda N. et al., Nippon Rinsho 1999 August, 57(8):1810), myxedema, idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 August, 57(8):1759), autoimmune reproductive diseases, ovarian diseases, ovarian autoimmunity (Garza KM. et al., J Reprod Immunol 1998 February, 37(2):87), autoimmune antisperm infertility (Diekman AB. et al., Am J Reprod Immunol. 2000 March, 43(3):134), recurrent fetal death (Tincani A. et al., Lupus 1998, 7 Suppl 2:S107-9), neurological diseases, neurological diseases, neurological autoimmune diseases, multiple sclerosis (Cross A H.ら, J Neuroimmunol January 1, 2001, 112(1-2):1), Oron L. disease (Oron L.et al., J Neural Transm Suppl 1997,49:77), myasthenia gravis (Infante AJ. and Kraig E, Int Rev Immunol 1999,18(1-2):83), motor neuropathy (Kornberg AJ.J Clin Neurosci.2000 May,7(3):191), Guillain-Barre syndrome, neuropathy and autoimmune neuropathy (Kusunoki S.Am J Med Sci.2000 April,319(4):234), myasthenic disorders, Lambert-Eaton myasthenic syndrome (Takamori M.Am J Med Sci. April 2000;319(4):204), paraneoplastic neurological disorders, cerebellar atrophy, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff-man syndrome, cerebellar atrophy, progressive cerebellar atrophy, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham's chorea, Gilles de la Tourette's syndrome, polyendocrinopathy, autoimmune polyendocrinopathy (Antoine JC. and Honnorat J. Rev Neurol (Paris) January 2000;156(1):23), neuropathy, immunodeficiency neuropathy (Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999;50:419), neuromyotonia, acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al., Ann NY Acad Sci. 1998 May 13, 841:482), cardiovascular diseases, cardiovascular autoimmune diseases, atherosclerosis (Matsuura E. et al., Lupus. 1998, 7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998, 7 Suppl 2:S132), thrombosis (Tincani A. et al., Lupus. 1998, 7 Suppl 2:S107-9), granulomatosis, Wegener's granulomatosis, arteritis, Takayasu's arteritis and Kawasaki disease (Praprotnik S. et al., Wien Klin Wochenschr. 2000, 8 Aug 25, 112(15-16):660), anti-factor VIII autoimmune diseases (Lacroix-Desmazes S. et al., Semin Thromb Hemost.2000,26(2):157), vasculitis, necrotizing small vessel vasculitis, microscopic polyangiitis, Churg-Strauss syndrome, glomerulonephritis, partial immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel LH.Ann Med Interne(Paris).2000 May,151(3):178), antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis 1999,14(4):171), heart failure, agonist-like β-adrenergic receptor antibodies in heart failure (Wallukat G. et al., Am J Cardiol.1999 Jun 17,83(12A):75H), thrombocytopenic purpura (Moccia F.Ann Ital Med Int. April-June 1999, 14(2):114), hemolytic anemia, autoimmune hemolytic anemia (Efremov DG. et al., Leuk Lymphoma January 1998, 28(3-4):285), gastrointestinal diseases, autoimmune diseases of the gastrointestinal tract, intestinal diseases, chronic inflammatory bowel diseases (Garcia Herola A. et al., Gastroenterol Hepatol. January 2000, 23(1):16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah January 16, 2000, 138(2):122), autoimmune diseases of the muscular system, myositis, autoimmune myositis, Sjögren's syndrome ((Feist E. et al., Int Arch Allergy Immunol. September 2000, 123(1):92), Smooth Muscle Autoimmune Diseases (Zauli D. et al., Biomed Pharmacother January 1999, 53(5-6):234), liver disease, liver autoimmune disease, autoimmune hepatitis (Manns MP. J Hepatol August 2000, 33(2):326) and primary biliary cirrhosis (Strassburg CP. et al., Eur J Gastroenterol Hepatol. June 1999, 11(6):595).

[0140] Type IV is also a T cell-mediated hypersensitivity disorder, including leukoencephalopathy, joint leukoencephalopathy (Tisch R, McDevitt HO. Proc Natl Acad Sci USA 1994 January 18, 91(2):437), systemic disease, systemic autoimmune disease, systemic lupus erythematosus (Datta SK., Lupus 1998, 7(9):591), gland disease, gland autoimmune disease, pancreatic disease, pancreatic autoimmune disease, type I diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647), thyroid disease, autoimmune thyroid disease, Graves' disease (Sakata S., Mol Cell Endocrinol 1998, 7(9):591). 1993, March, 92(1):77), ovarian disease (Garza KM., J Reprod Immunol 1998 February, 37(2):87), prostatitis, autoimmune prostatitis (Alexander RB., Urology 1997 December, 50(6):893), polyglandular syndrome, autoimmune polyglandular syndrome, autoimmune polyglandular syndrome type I (Hara T., Blood. 1991 March 1, 77(5):1127), neurological disease, autoimmune neurological disease, multiple sclerosis, neuritis, optica neuritis (Soderstrom M., Neurol Neurosurg Psychiatry 1994 May, 57(5):544), myasthenia gravis (Oshima M., Eur J Immunol 1990 December, 20(12):2563), Stephan syndrome (Hiemstra HS., Proc Natl Acad Sci USA 2001 March 27, 98(7):3988), cardiovascular disease, cardiac autoimmunity in schizophrenia (Cunha-Neto E., J Clin Invest 1996 October 15, 98(8):1709), autoimmune thrombocytopenic purpura (Semple JW., Blood May 15, 1996, 87(10):4245), anti-Halper Trinidad autoimmunity (Caporossi AP., Viral Immunol 1998, 11(1):9), hemolytic anemia (Sallah S., Ann Hematol 1997 March, 74(3):139), liver disease, autoimmune liver disease, hepatitis, chronic active hepatitis (Franco A.et al., Clin Immunol Immunopathol March 1990, 54(3):382), biliary cirrhosis, primary biliary cirrhosis (Jones DE. Clin Sci(Colch) November 1996, 91(5):551), renal disease, renal autoimmune disease, nephritis, interstitial nephritis (Kelly CJ.J Am Soc Nephrol Aug. 1990, 1(2):140), connective tissue diseases, ear diseases, autoimmune connective tissue diseases, autoimmune ear diseases (Yoo TJ. et al., Cell Immunol Aug. 1994, 157(1):249), diseases of the inner ear (Gloddek B. et al., Ann NY Acad Sci 1997 Dec. 29, 830:266), skin disease, dermatological disease, skin disease, bullous skin disease, pemphigus vulgaris, bullous pemphigoid, and pemphigus foliaceus.

[0141] Examples of delayed-type hypersensitivity include, but are not limited to, contact dermatitis and drug rash.

[0142] Examples of types of T lymphocytes that mediate hypersensitivity include, but are not limited to, helper T lymphocytes and cytotoxic T lymphocytes.

[0143] Examples of helper T lymphocyte-mediated hypersensitivity include T h 1 Lymphocyte-mediated hypersensitivity and T h 2 Lymphocyte-mediated hypersensitivity.

[0144] autoimmune diseases These include, but are not limited to, cardiovascular diseases, rheumatic diseases, glandular diseases, gastrointestinal diseases, skin diseases, liver diseases, neurological diseases, muscular diseases, renal diseases, diseases related to reproduction, connective tissue diseases and systemic diseases.

[0145] Examples of autoimmune cardiovascular diseases include atherosclerosis (Matsuura E. et al., Lupus. 1998, 7 Suppl 2: S135), myocardial infarction (Vaarala O., Lupus. 1998, 7 Suppl 2: S132), thrombosis (Tincani A. et al., Lupus. 1998, 7 Suppl 2: S107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki disease (Praprotnik S. et al., Wien Klin Wochenschr. 2000, 25 Aug., 112(15-16):660), anti-factor VIII autoimmune diseases (Lacroix-Desmazes S. et al., Semin Thromb. Hemost. 2000, 26(2):157), necrotizing small angiitis, microscopic polyangiitis, Churg-Strauss syndrome, partial immune focal necrotizing glomerulonephritis, and crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). May 2000, 151(3):178), antiphospholipid antibody syndrome (Flamholz R. et al., J Clin Apheresis 1999, 14(4):171), antibody-induced heart failure (Wallukat G. et al., Am J Cardiol. June 17, 1999, 83(12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. April-June 1999, 14(2):114, Semple J.W. et al., Blood 1996 May 15;87(10):4245), autoimmune hemolytic anemia (Efremov DG. et al., Leuk Lymphoma 1998 January 28(3-4):285, (Sallah S. et al., Ann Hematol 1997 March 74(3):139), cardiac autoimmunity in Chagas disease (Cunha-Neto E. et al., J Clin Invest 1996 October 15;98(8):1709) and anti-helper T lymphocyte autoimmunity (Caporossi AP. et al., Viral Immunol 1998,11(1):9).

[0146] Examples of autoimmune rheumatic diseases include, but are not limited to, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 July; 15(3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units SA 1994 January 18; 91(2):437) and ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3(3):189).

[0147] Examples of autoimmune glandular diseases include, but are not limited to, pancreatic disease, type I diabetes, thyroid disease, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune antizoospermia, autoimmune prostatitis, and autoimmune polyglandular syndrome type I. Diseases include autoimmune pancreatic disease, type I diabetes (Castano L. and Eisenbarth GS.Ann.Rev.Immunol.8:647, Zimmet P.Diabetes Res Clin Pract October 1996, 34 Supplement: S125), autoimmune thyroid disease, Graves' disease (hyperthyroidism) (Orgiazzi J. Endocrinol Metab Clin North Am June 2, 2000, 9(2):339, Sakata S. et al., Mol Cell Endocrinol March 1993, 92(1):77), Spontaneous autoimmune thyroiditis (Braley-Mullen H. and J Immunol December 15, 2000, 165(12):7262), Hashimoto's thyroiditis (Toyoda N. et al., Nippon Rinsho 1999 Aug;57(8):1810), idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57(8):1759), ovarian autoimmunity (Garza KM. et al. J Reprod Immunol. 1998 Feb;37(2):87), autoimmune antizoospermia (Diekman AB. et al. Am J Reprod Immunol. 2000 Mar;43(3):134), autoimmune prostatitis (Alexander RB. et al. Urology 1997 Dec;50(6):893) and type I autoimmune polyglandular syndrome (Hara T. et al. Blood. 1991 Mar;77(5):1127).

[0148] Examples of autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory bowel disease (Garcia Herola A. et al., Gastroenterol Hepatol. 2000 January 23(1):16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 January 16, 138(2):122), colitis, ileitis, and Crohn's disease.

[0149] Examples of autoimmune skin diseases include, but are not limited to, autoimmune bullous skin diseases such as pemphigus vulgaris, bullous pemphigoid, and pemphigus foliaceus.

[0150] Examples of autoimmune liver diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 March; 54(3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 November; 91(5):551; Strassburg CP. et al., Eur J Gastroenterol Hepatol. 1999 June; 11(6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 August; 33(2):326).

[0151] Examples of autoimmune neurological diseases include multiple sclerosis (Cross AH. et al., J Neuroimmunol 2001 Jan 112(1-2):1), Alzheimer's disease (Oron L. et al., J Neural Transm Suppl 1997, 49:77), myasthenia gravis (Infante AJ. and Kraig E., Int Rev Immunol 1999, 18(1-2):83; Oshima M. et al., Eur J Immunol 1990 Dec 20(12):2563), neuropathy, motor neuropathy (Kornberg AJ. J Clin Neurosci. 2000 May 7(3):191), Guillain-Barre syndrome and autoimmune neuropathy (Kusunoki S. Am J Med Sci. 2000 Apr;319(4):234), myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr;319(4):204), paraneoplastic neurological disorders, cerebellar atrophy, paraneoplastic cerebellar atrophy, and stiff-man syndrome (Hiemstra HS. et al. Proc Natl Acad Sci units SA 2001 Mar 27;98(7):3988), non-paraneoplastic stiff-man syndrome, progressive cerebellar atrophy, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham's chorea, Gilles de la Tourette's syndrome, and autoimmune polyendocrinopathy (Antoine JC. and Honnorat J. Rev Neurol (Paris) January 2000, 156(1):23), immunodeficiency neuropathy (Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999, 50:419), acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al., Ann NY Acad Sci. May 13, 1998, 841 482), neuritis, optic neuritis (Soderstrom M. et al., Neurol Neurosurg Psychiatry May 1994, 57(5):544) and neurodegenerative diseases.

[0152] Examples of autoimmune muscle diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren's syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sept. 123(1):92) and smooth muscle autoimmune diseases (Zauli D. et al., Biomed Pharmacother 1999 Jan. 53(5-6):234).

[0153] Examples of autoimmune renal diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;1(2):140).

[0154] Examples of autoimmune diseases related to reproduction include, but are not limited to, recurrent fetal death (Tincani A. et al., Lupus 1998, 7 Suppl 2:S107-9).

[0155] Examples of autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et al., Cell Immunol 1994 Aug; 157(1):249) and autoimmune diseases of the inner ear (Gloddek B. et al., Ann NY Acad Sci 1997 Dec. 29; 830:266).

[0156] Examples of autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998, 17(1-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol. 1999 Mar. 6(2):156; Chan OT. et al., Immunol Rev 1999 Jul. 169 107).

[0157] According to certain embodiments, the autoimmune disease is selected from the group consisting of multiple sclerosis, myasthenia gravis, Guillain-Barre syndrome (antiphospholipid syndrome), systemic erythromatosis, Behcet's syndrome, Sjogren's syndrome, rheumatoid arthritis, Hashimoto's / hypothyroidism, primary biliary cirrhosis, mixed connective tissue disease, chronic active hepatitis, Graves' / hyperthyroidism, scleroderma, chronic idiopathic thrombocytopenic purpura, diabetic neuropathy, and septic shock (see, e.g., Schneider A. et al., J Biol Chem. 279:55833-9 (2004)).

[0158] infectious disease Examples of infectious diseases include, but are not limited to, chronic infections, subacute infections, acute infections, viral diseases, bacterial diseases, protozoal diseases, parasitic diseases, fungal diseases, mycoplasmal diseases, and prion diseases.

[0159] Transplant Rejection Disorders Examples of diseases associated with transplantation of a graft include, but are not limited to, graft rejection, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection, and graft-versus-host disease.

[0160] Allergic diseases Examples of allergic disorders include, but are not limited to, asthma, hives, urticaria, pollen allergies, dust mite allergies, poison allergies, cosmetic allergies, latex allergies, chemical allergies, drug allergies, insect bite allergies, animal dander allergies, stinging plant allergies, poison ivy allergies, and food allergies.

[0161] Cancerous diseases Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Specific examples of cancerous diseases include, but are not limited to, myeloid leukemia, such as chronic myeloid leukemia. Acute myeloid leukemia with maturation. Acute promyelocytic leukemia, acute nonlymphocytic leukemia with basophilia, acute monocytic leukemia. Lymphocytic leukemia, such as acute myelomonocytic leukemia with eosinophilia, malignant lymphoma, such as Birkitt's non-Hodgkin's lymphoma, and acute lymphoblastic leukemia. Chronic lymphocytic leukemia, solid tumors, benign meningiomas, mixed tumors of the salivary glands, myeloproliferative disorders such as colon adenoma, small cell lung cancer, kidney cancer, uterine cancer, prostate cancer, bladder cancer, ovarian cancer, colon cancer, sarcoma, liposarcoma, myxoid carcinoma, synovial sarcoma, rhabdomyosarcoma (alveolar), extraskeletal myxoid chondrosarcoma, adenocarcinomas such as Ewing's tumor, and others include testicular and ovarian dysgerminoma, retinoblastoma, Wilms' tumor, neuroblastoma, malignant melanoma, mesothelioma, breast cancer, skin cancer, prostate cancer, and ovarian cancer.

[0162] According to some aspects of the invention, the disease or condition is a SHANK3-associated disease.

[0163] As used herein, the term "SHANK3" (also known as SH3 and multiple ankyrin repeat domains 3 and proline-rich synapse-associated protein 2 (ProSAP2)) refers to the expression product, e.g., RNA or protein, of the SHANK3 gene (Gene ID 85358). This gene encodes a protein that contains five interaction domains or motifs, including ankyrin repeats domain (ANK), a src3 domain (SH3), a proline-rich domain, a PDZ domain, and a sterile α motif domain (SAM).

[0164] According to certain embodiments, the SHANK3 is human SHANK3 as provided in the following catalog numbers: NM_001080420, NM_001372044, NP_277052.

[0165] As used herein, the term "SHANK3-associated disease" refers to a disease whose onset and / or progression is associated with impaired SHANK3 function (e.g., due to mutations). A non-limiting example of such a disease is Phelan-McDermid syndrome.

[0166] The inventors have demonstrated gender-related differences in some, but not all, aspects of the effects of the polypeptides and active agents disclosed herein. Thus, in some embodiments, the subject is male. In other embodiments, the subject is female.

[0167] In some embodiments, treatment with the methods, compositions or active ingredients of the articles of manufacture of the invention may have greater efficacy in certain age groups, e.g., younger subjects in which neurodevelopmental processes are affected, and conversely, older subjects in which neurodegenerative or aging processes are affected. Thus, in some embodiments, the subject is younger than 18 years of age, i.e., 0-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years of age, but less than 18 years of age. In other embodiments, the subject is over 60 years of age, i.e., in the ranges 61-65 years, 64-70 years, 69-75 years, 74-80 years, 79-85 years, 84-90 years, 89-95 years, 94-100 years, and over 100 years of age. It will be understood that age ranges include all intervening ages.

[0168] As used herein, the term "cognitive disorder" encompasses both intellectual disability and cognitive impairment (typically associated with a psychiatric disorder or neurodegenerative disease).

[0169] As used herein, the term "intellectual disability (ID)", also known as general learning disability or mental retardation (MR), refers to a generalized neurodevelopmental disorder characterized by significantly impaired intellectual and adaptive functioning.

[0170] As used herein, the term "Activity-Dependent Neuroprotective Factor (ADNF)" refers to ADNF III (also known as ADNP) and / or ADNF I.

[0171] As used herein, the term "ADNF polypeptide" refers to the amino acid sequence of human ADNF III and / or ADNF I, or a functional homologue thereof, having at least one activity of ADNF III or ADNF I, as further described below. According to certain embodiments, the phrase "ADNF polypeptide" refers to a mixture of ADNF III and ADNF I polypeptides.

[0172] As used herein, the phrase "functional homolog" refers to fragments, naturally occurring or synthetically / recombinantly produced homologs, non-human homologs, allelic or polymorphic variants, amino acid sequences including conservative and non-conservative amino acid substitutions deletions or additions, analogs, lipophilic variants and / or chemically modified variants that maintain at least one of the activities of the full-length protein (e.g., neurotrophic / neuroprotective activity, binding to EB1 and / or EB3, binding to the SH3 domain), as further described below.

[0173] As used herein, the terms "polypeptide", "peptide" or "amino acid sequence" (which are used interchangeably herein) include native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically synthetically synthesized peptides), as well as peptoids and semipeptoids that are peptide analogs, which may have modifications that, for example, make the peptide more stable in the body or more permeable in cells. Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications, backbone modifications, and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA Ramsden Gd, Chapter 17.2, F. Choplin Pergamon Press (1992), which are incorporated herein by reference as if fully set forth herein. Further details on this subject are provided below.

[0174] The peptide bond (-CO-NH-) in a peptide can be, for example, an N-methylated amide bond (-N(CH3)-CO-), an ester bond (-C(=O)-O-), a ketomethylene bond (-CO-CH2-), a sulfinylmethylene bond (-S(=O)-CH2-), an α-aza bond (-NH-N(R)-CO-) (where R is any alkyl (e.g., methyl)), an amine bond (-CH2-NH-), a sulfide bond (-CH2- S-), ethylene bond (-CH2-CH2-), hydroxyethylene bond (-CH(OH)-CH2-), thioamide bond (-CS-NH-), olefinic double bond (-CH=CH-), fluorinated olefinic double bond (-CF=CH-), retroamide bond (-NH-CO-), peptide derivatives (-N(R)-CH2-CO-), where R is a naturally occurring "normal" side chain on a carbon atom.

[0175] These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) bonds at the same time.

[0176] The natural aromatic amino acids, Trp, Tyr and Phe may be replaced by unnatural aromatic amino acids such as 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe or O-methyl-Tyr.

[0177] Polypeptides of some embodiments of the invention may also contain one or more modified amino acids or one or more non-amino acid monomers (eg, fatty acids, complex carbohydrates, etc.).

[0178] The term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids, such as those amino acids that are frequently post-translationally modified in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine, as well as other unusual amino acids, including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine. Additionally, the term "amino acid" includes both D- and L-amino acids.

[0179] According to certain embodiments, the polypeptide comprises at least one D-amino acid.

[0180] According to certain embodiments, the polypeptide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 8 D-amino acids.

[0181] According to certain embodiments, all polypeptide amino acids are D-amino acids.

[0182] Tables 1 and 2 below list naturally occurring amino acids (Table 2) and non-conventional or modified amino acids (eg, synthetic, Table 3) that may be used in some embodiments of the invention.

[0183] [Table 1]

[0184] [Table 2] JPEG2024511616000004.jpg217159

[0185] Amino acid substitutions in polypeptides of some embodiments of the present invention may be either conservative or non-conservative.

[0186] The term "conservative substitution" as used herein refers to the replacement of an amino acid present in a natural sequence in a peptide with a naturally occurring or non-naturally occurring amino acid or a peptidomimetic having similar stereochemical properties. If the side chain of the natural amino acid being substituted is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid, or a peptidomimetic moiety that is polar or hydrophobic (in addition to having the same stereochemical properties as the side chain of the substituted amino acid).

[0187] Since naturally occurring amino acids are typically classified according to their properties, conservative substitutions with naturally occurring amino acids can be readily determined, taking into account the fact that, in accordance with the present invention, the replacement of a charged amino acid with a stereochemically similar uncharged amino acid is considered a conservative substitution.

[0188] It is also possible to use amino acid analogues (synthetic amino acids) well known in the art to generate conservative substitutions with non-naturally occurring amino acids. Peptide mimetics of naturally occurring amino acids are well described in the literature well known to those skilled in the art.

[0189] When affecting a conservative substitution, the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.

[0190] Conservative substitution tables providing functionally similar amino acids are well known in the art. Guidance concerning which amino acid changes are likely to be phenotypically silent can also be found in Bowie et al., 1990, Science 247:1306 1310.

[0191] The phrase "non-conservative substitution" as used herein refers to the replacement of an amino acid present in a parent sequence with another naturally occurring amino acid or a non-naturally occurring amino acid having different electrochemical and / or stereochemical properties. Thus, the side chain of the substituted amino acid may be significantly larger (or smaller) than the side chain of the natural amino acid being replaced, and / or may have a functional group with significantly different electronic properties than the amino acid being replaced. Examples of this type of non-conservative substitution include the substitution of phenylalanine or cyclohexylmethylglycine for alanine, the substitution of isoleucine for glycine, or the substitution of -NH-CH[(-CH2)5-COOH]-CO- for aspartic acid. These non-conservative substitutions within the scope of the present invention still constitute peptides with neuroprotective properties.

[0192] The polypeptides of some embodiments of the present invention are preferably utilized in linear form, although it will be understood that cyclic forms of the peptides may also be utilized where cyclization does not significantly interfere with the characteristics of the peptide.

[0193] According to certain embodiments, since the polypeptides of the present invention are utilized in therapeutic applications that require the peptide to be in a soluble form, the polypeptides of some embodiments of the present invention contain one or more unnatural or natural polar amino acids, including, but not limited to, serine and threonine, which can increase the solubility of the peptide due to their hydroxyl-containing side chains.

[0194] According to certain embodiments, the polypeptide is less than 100, less than 50, less than 20, or less than 10 amino acids in length.

[0195] According to specific embodiments, the polypeptide is between 4 and 100, 4 and 50, 4 and 40, 4 and 20, 4 and 15, 4 and 10, 4 and 8, or 8 amino acids in length, each possibility representing a separate embodiment of the present invention.

[0196] According to certain embodiments, the polypeptide is at least 4, at least 5, at least 6, at least 7, at least 8 amino acids in length.

[0197] According to certain embodiments, the polypeptide is linked, either directly or via a spacer or linker, to a cell-penetrating moiety and / or a stabilizing moiety, such moieties being well known in the art and described in more detail below.

[0198] According to certain embodiments, the N-terminus and / or C-terminus of the polypeptides of some embodiments of the present invention may be protected by a functional group (i.e., an end-capping moiety). Examples of such functional groups can be found, for example, in Green et al., "Protective Groups in Organic Chemistry", (Wiley, 2.sup.nd ed. 1991), Harrison et al., "Compendium of Synthetic Organic Methods", Vols. 1-8 (John Wiley and Sons, 1971-1996), and Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference. Preferred protecting groups are those which increase the stability of the polypeptide and / or facilitate the transport of the compound bound thereto into cells, for example by decreasing the hydrophilicity and increasing the lipophilicity of the compound.

[0199] According to certain embodiments, the end-capping comprises an N-terminal end-capping.

[0200] Representative examples of N-terminal end-capping moieties include, but are not limited to, formyl, acetyl (also referred to herein as "Ac"), stearyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (also referred to herein as "Cbz"), tert-butoxycarbonyl (also referred to herein as "Boc"), trimethylsilyl (also referred to herein as "TMS"), 2-trimethylsilyl-ethanesulfonyl (also referred to herein as "SES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (also referred to herein as "Fmoc"), and nitro-veratryloxycarbonyl ("NVOC").

[0201] According to certain embodiments, the N-terminal end-capping comprises an acetyl.

[0202] According to certain embodiments, the N-terminal end-capping comprises stearyl (see, for example, Gozes I et al., Proc Natl Acad Sci US A. 1996 Jan. 9, 93(1):427-32).

[0203] According to certain embodiments, the end-capping comprises a C-terminal end-capping.

[0204] Representative examples of C-terminal end-capping moieties are those that typically result in acylation of a carboxy group at the C-terminus, and include, but are not limited to, benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers, allyl ethers, monomethoxytrityl and dimethoxytrityl. Alternatively, the -COOH group of the C-terminal end-capping may be modified to an amide group.

[0205] According to certain embodiments, the C-terminal end capping comprises an amide.

[0206] Other end-capping modifications of peptides include replacement of the amine and / or carboxyl with different moieties such as hydroxyl, thiol, halide, alkyl, aryl, alkoxy, aryloxy, and the like.

[0207] According to other particular embodiments of the invention, the polypeptide is conjugated to a non-proteinaceous moiety.

[0208] According to certain embodiments, the polypeptide and the attached non-proteinaceous moiety are covalently linked, either directly or via a spacer or linker.

[0209] The phrase "non-proteinaceous moiety" as used herein refers to a molecule that does not contain peptide-bound amino acids attached to the above polypeptide. According to certain embodiments, the non-proteinaceous moiety is a non-toxic moiety. Exemplary non-proteinaceous moieties that may be used according to the present teachings include, but are not limited to, drugs, chemicals, small molecules, polynucleotides, detectable moieties, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), poly(styrene comaleic anhydride (SMA), and divinyl ether and maleic anhydride copolymer (DIVEMA). According to certain embodiments of the present invention, the non-proteinaceous moiety comprises polyethylene glycol (PEG).

[0210] Such molecules are very stable (resisting in vivo proteolytic activity, presumably due to steric hindrance imparted by the nonproteinaceous moieties) and may be produced using inexpensive and highly efficient conventional solid-phase synthetic methods, as described further below, however, it will be appreciated that recombinant techniques may still be used in which the recombinant peptide product is subjected to in vitro modifications (e.g., PEGylation, as described further below).

[0211] Bioconjugation of peptide amino acid sequences with PEG (i.e., PEGylation) can be carried out using PEG derivatives such as N-hydroxysuccinimide (NHS) esters of PEG carboxylic acids, monomethoxy PEG2-NHS, succinimidyl esters of carboxymethylated PEG (SCM-PEG), benzotriazole carbonate derivatives of PEG, glycidyl ethers of PEG, PEG p-nitrophenyl carbonate (PEG-NPC, e.g., methoxy PEG-NPC), PEG aldehydes, PEG-orthopyridyl-disulfides, carbonyldiimidazole-activated PEG, PEG-thiols, and PEG-maleimides. Such PEG derivatives are commercially available in a variety of molecular weights (see, e.g., Catalog, Polyethylene Glycol and Derivatives, 2000 (Shearwater Polymers, Inc., Huntsville, AL)). Many of the above derivatives are available in monofunctional monomethoxy PEG (mPEG) form, if desired. In general, the PEG attached to the peptides of some embodiments of the invention should range in molecular weight (MW) from a few hundred daltons to about 100 kDa (e.g., 3-30 kDa). Larger MW PEGs can be used, but may result in some loss of yield of the PEGylated polypeptide. The purity of the larger PEG molecules should also be taken into consideration, as it may be difficult to obtain larger MW PEGs of as high a purity as that obtained for lower MW PEGs. It is preferred to use PEGs of at least 85% purity, and more preferred to use PEGs of at least 90% purity, 95% purity, or higher.PEGylation of molecules is further discussed, for example, in Chapter 15 of Hermanson, Bioconjugate Techniques, Academic Press San Diego, CA (1996), and Zalipsky et al., "Succinimidyl Carbonates of Polyethylene Glycol," in Polymeric Drugs and Drug Delivery Systems, edited by Dunn and Ottenbrite, American Chemical Society, Washington, DC (1991).

[0212] Conveniently, PEG can be attached to a selected position in a peptide by site-directed mutagenesis, as long as the activity of the conjugate is retained. The target of PEGylation can be any cysteine ​​residue at the N-terminus or C-terminus of the peptide sequence. Additionally or alternatively, other cysteine ​​residues can be added to the peptide amino acid sequence (e.g., at the N-terminus or C-terminus) and thereby serve as targets for PEGylation. Computer analysis can be performed to select preferred positions for mutagenesis without compromising activity.

[0213] Various conjugation chemistries of activated PEG can be used, such as PEG-maleimide, PEG-vinylsulfone (VS), PEG-acrylate (AC), PEG-orthopyridyl disulfide, etc. Methods for preparing activated PEG molecules are well known in the art. For example, PEG-VS can be prepared by reacting a dichloromethane (DCM) solution of PEG-OH with NaH and then with divinylsulfone under argon (molar ratio: OH 1:NaH 5:divinylsulfone 50 at 0.2 grams PEG / mL DCM). PEG-AC is made by reacting a DCM solution of PEG-OH with acryloyl chloride and triethylamine under argon (molar ratio: OH 1:acryloyl chloride 1.5:triethylamine 2 at 0.2 grams PEG / mL DCM). Such chemical groups can be attached to linearized 2-arm, 4-arm, or 8-arm PEG molecules.

[0214] The resulting conjugated molecules (e.g., PEGylated or PVP-conjugated peptides) are isolated, purified, and characterized using, for example, High-Performance Liquid Chromatography (HPLC) as well as biological assays.

[0215] The polypeptides and compositions of matter of the invention may be attached (either covalently or non-covalently) to a permeable moiety.

[0216] According to other specific embodiments, the polypeptide is not bound to a heterologous permeable moiety.Thus, for example, the ADNF polypeptide NAP (SEQ ID NO:2) is bioavailable by endocytic cell motility (see, for example, Ivashko-Pachima Y, Gozes IJ Mol Neurosci. July 2020, 70(7):993-998), and thus is itself a cell-permeable peptide.

[0217] As used herein, the phrase "permeable moiety" refers to an agent that enhances the movement across a cell membrane of either an attached polypeptide or a composition of matter comprising same.

[0218] According to one embodiment, the permeabilizing moiety is a peptide and is attached (directly or indirectly) to the polypeptide via a peptide bond.

[0219] Typically, peptide permeable moieties have an amino acid composition that contains a high relative abundance of positively charged amino acids such as lysine or arginine, or have a sequence that contains an alternating pattern of polar / charged and non-polar hydrophobic amino acids.

[0220] As a non-limiting example, a Cell Penetrating Peptide (CPP) sequence may be used to enhance intracellular penetration, however, the disclosure is not so limited and any suitable penetrating agent may be used, as known to those of skill in the art.

[0221] Cell-penetrating peptides (CPPs) are short peptides (≦40 amino acids) that have the ability to access the interior of almost any cell. They are highly cationic and are usually rich in arginine and lysine amino acids. They have the exceptional property of carrying a wide variety of covalently and non-covalently conjugated cargos, such as proteins, oligonucleotides, and even 200 nm liposomes, into cells. Thus, according to a further exemplary embodiment, CPPs can be used to transport ADNP polypeptides to the interior of cells.

[0222] TAT (transcriptional activator from HIV-1), pAntp (penetratin, also called Drosophila antennapedia homeodomain transcription factor), and VP22 (from herpes simplex virus) are non-limiting examples of CPPs that may enter cells in a non-toxic and efficient manner and may be suitable for use in some embodiments of the present invention. Protocols for producing CPP-cargo conjugates and for infecting cells with such conjugates can be found, for example, in L Theodore et al., The Journal of Neuroscience, (1995) 15(11):7158-7167, Fawell S et al., Proc Natl Acad Sci USA, (1994) 91:664-668, and Bian et al., Circulation Research. (2007) 100:1626-1633.

[0223] According to another exemplary embodiment, the polypeptide may be incorporated into a microparticulated delivery vehicle (e.g., a liposome, or a micro- or microparticle) by any of the methods well known in the art [e.g., Liposome Technology, Vol. II, Incorporation of Drugs, Proteins, and Genetic Material, CRC Press, Monkkonen, J. et al., 1994, J. Drug Target, 2:299-308; Monkkonen, J. et al., 1993, Calcif. Tissue Int., 53:139-145; Lasic D D., Liposomes Technology Inc., Elsevier, 1993, 63-105. (chapter 3); Winterhalter M, Lasic DD, Chem Phys Lipids, September 1993, 64(1-3):35-43].

[0224] Liposomes include any synthetic (i.e., non-naturally occurring) structure composed of a lipid bilayer enclosing a volume. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. Liposomes can be of different sizes, contain low or high pH, ​​and can be of different charges.

[0225] Polypeptides of some embodiments of the present invention may be synthesized by any technique well known to those skilled in the art of peptide synthesis, including, but not limited to, solid phase and recombinant techniques.

[0226] For solid phase peptide synthesis, overviews of many techniques can be found in JM Stewart and JD Young, Solid Phase Peptide Synthesis, WH Freeman Co. (San Francisco), 1963, and in J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 1973. For classical solution synthesis, see G. Schroder and K. Lupke, The Peptides, vol. 1, Academic Press (New York), 1965.

[0227] In general, these methods involve the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain. Usually, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence with the complementary (amino or carboxyl) group suitably protected under suitable conditions to form an amide bond. The protecting group is then removed from this newly added amino acid residue, and then the next amino acid (suitably protected) is added, and so on. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support) are removed sequentially or simultaneously to obtain the final peptide compound. Simple modifications of this basic procedure make it possible to add more than one amino acid at a time to the growing chain, for example, by coupling a protected tripeptide with a suitably protected dipeptide (under conditions that do not racemize the chiral centers) to form, after deprotection, a pentapeptide, etc. Further description of peptide synthesis is disclosed in US Pat. No. 6,472,505.

[0228] Large scale peptide synthesis is described in Andersson Biopolymers 2000, 55(3):227-50. Certain embodiments of the present invention contemplate the use of combination therapeutic / prophylactic agents comprising a polypeptide and a therapeutic agent other than the polypeptides disclosed herein.

[0229] Thus, according to certain embodiments, the polypeptides disclosed herein may be provided to an individual together with additional active agents to achieve improved therapeutic or prophylactic effects compared to treatment with each agent alone. Thus, the polypeptides may be administered alone or together with other established or experimental therapeutic regimens to treat or prevent diseases associated with evoked potentials and / or language disorders, autism spectrum disorders and intellectual disabilities, Alzheimer's disease, autism spectrum disorders, neurodegenerative diseases, cognitive disorders, psychiatric disorders and cytoskeletal disorders, as detailed herein. In such treatments, measures are taken (e.g., dosing and selection of complementary agents) to minimize or eliminate adverse side effects that may be associated with combination therapy.

[0230] Non-limiting examples of ADNF polypeptides that may be used in certain embodiments of the invention are described, for example, in PCT International Publication Nos. WO 1992 / 018140, WO 9611948, WO 98 / 35042, WO 0027875, WO 00 / 53217, WO 01 / 12654, WO 01 / 92333, WO 2004 / 080957, WO 2006 / 099739, WO 2007 / 096859, WO 2008 / 0844, and the like. 8, WO2011 / 021186, WO / 2009 / 026687, WO2010 / 075635, WO2011 / 083461, WO2011 / 099011, WO2013 / 171595, WO2017 / 130190, WO2004 / 060309, WO2003022226, and U.S. Patent Nos. 5,767,240, 6,174,862, 6,613,740, and 8,586,548, each of which is incorporated herein by reference in its entirety and further described below.

[0231] According to a particular embodiment, the ADNF is ADNF III.

[0232] "ADNF III" is also known as ADNP (activity-dependent neuroprotective factor) and refers to a polypeptide encoded by the ADNP gene (gene ID 23394). According to certain embodiments, the ADNF III is human ADNF III. Full-length human ADNF III (ADNP) has a predicted molecular weight of 123,562.8 Da (>1000 amino acid residues) and a theoretical pI of about 6.97. The human ADNF III gene is localized to chromosome 20q13.13-13.2, a region associated with cognitive function. Representative full-length amino acid and nucleic acid sequences of ADNF III can be found in PCT International Publication Nos. WO98 / 35042, WO00 / 27875, U.S. Patent Nos. 6,613,740 and 6,649,411. According to certain embodiments, the ADNF III amino acid sequence comprises SEQ ID NO:1.

[0233] The ADNF III polypeptides described herein have at least one activity of full-length ADNF III, e.g., neurotrophic / neuroprotective activity as measured in an in vitro cortical neuronal culture assay, binding to EB1 and / or EB3, and binding to the SH3 domain.

[0234] Assays for testing neurotrophic / neuroprotective activity are well known in the art and include, but are not limited to, for in vitro cortical neuronal culture assays, see, for example, Hill et al., Brain Res. 603:222:233 (1993); Brenneman and Gozes, J. Clin. Invest. 97:2299-2307 (1996); Gozes et al., Proc. Natl. Acad. ScL USA 93, 427-432 (1996).

[0235] Assays for testing binding are well known in the art and include, but are not limited to, flow cytometry, BiaCore, bio-layer interferometry Blitz® assays, and HPLC.

[0236] Non-limiting examples of ADNF III polypeptides that may be used in certain embodiments of the present invention are provided in Table 3 below.

[0237] According to certain embodiments, the ADNF III polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity or homology to any of SEQ ID NOs:1-22.

[0238] As used herein, "identity" or "sequence identity" refers to global identity, i.e., identity over the entire amino acid or nucleic acid sequence disclosed herein, but not over a portion thereof.

[0239] Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm, such as Blast, ClustalW, and MUSCLE.

[0240] According to a particular embodiment, the ADNF is ADNF I.

[0241] "ADNF I" is It refers to the activity-dependent neurotrophic factor described in Gozes I, Brenneman DE. J Mol Neurosci. Winter 1996, 7(4):235-44; Brenneman DE, Gozes I J Clin Invest. May 15, 1996, 97(10):2299-307; and Brenneman DE et al., J Pharmacol Exp Ther. May 1998, 285(2):619-27, the contents of each of which are incorporated herein by reference in their entirety. According to certain embodiments, the ADNF I is human ADNF I. Full-length human ADNF I has a predicted molecular weight of about 14,000 Da at 8.3±0.25 pi. According to certain embodiments, the ADNF I amino acid sequence comprises either SEQ ID NO:24 or SEQ ID NO:45.

[0242] The ADNF I polypeptides described herein have at least one activity of full-length ADNF I, eg, neurotrophic / neuroprotective activity as measured in an in vitro cortical neuronal culture assay, binding to EB1 and EB3.

[0243] Non-limiting examples of ADNF I polypeptides that may be used in certain embodiments of the invention are provided in Table 3 below.

[0244] According to certain embodiments, the ADNF I polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity or homology to any of SEQ ID NOs:24-48.

[0245] [Table 3] JPEG2024511616000006.jpg155158

[0246] In further embodiments, the polypeptide comprises an active core site comprising the amino acid sequence of NAPVSIPQ (SEQ ID NO:2) or SALLRSIPA (SEQ ID NO:24), or a conservatively modified variant (e.g., deletion, addition, or substitution of one or more amino acids) or a chemically modified variant thereof, and has neurotrophic / neuroprotective activity as measured in an in vitro cortical neuronal cell culture assay as described. The ADNF polypeptide may be derived from an ADNF I polypeptide, an ADNF III polypeptide, an allele thereof, a polymorphic variant, an analog, an interspecies homolog, any subsequence thereof, or a lipophilic variant that exhibits neuroprotective / neurotrophic activity on neurons derived from the central nervous system, for example, either in vitro or in vivo. ADNF-related neuroprotective peptides may range as short as 4-8 amino acids, and may have, for example, 8-20, 8-50, 10-100, or about 200, 500 or more amino acids. One non-limiting example of a mutant ADNP-related neuroprotective peptide is the four amino acid peptide of SKIP (SEQ ID NO:21), see Amram et al., Sexual Divergence in Microtubule Function: The Novel Intranasal Microtubule Targeting SKIP Normalizes Axonal Transport and Enhances Memory. Mol Psychiatry, 2016, 21:1467-76. Further examples include, but are not limited to, all D-amino acid derivatives of NAPVSIPQ (SEQ ID NO:13) and SALLRSIPA (SEQ ID NO:36).

[0247] Thus, according to a further aspect of the invention, the polypeptide has the formula 1 ) x -Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-(R 2 ) y (SEQ ID NO: 49), wherein R 1R is an amino acid sequence comprising from 1 to about 40 amino acids, each amino acid being independently selected from the group consisting of naturally occurring amino acids and amino acid analogs. 2 is an amino acid sequence comprising from 1 to about 40 amino acids, where each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs, and x and y are independently selected and equal to 0 or 1. In a further embodiment, the core amino acid sequence "Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln" of SEQ ID NO:49 ("Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln" is identical to SEQ ID NO:2) is replaced by an analog of SEQ ID NO:2.

[0248] According to a further aspect of the invention, the polypeptide has the formula 1 ) x -Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-(R 2 ) y (SEQ ID NO:50), wherein R 1 R is an amino acid sequence comprising from 1 to about 40 amino acids, each amino acid being independently selected from the group consisting of naturally occurring amino acids and amino acid analogs. 2 is an amino acid sequence comprising from 1 to about 40 amino acids, where each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs, and x and y are independently selected and equal to 0 or 1. In a further embodiment, the amino acid sequence "Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala" of SEQ ID NO:50 ("Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala" is identical to SEQ ID NO:24) is replaced by an analog of SEQ ID NO:24.

[0249] In some embodiments, the ADNF polypeptide is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-22. In other embodiments, the ADNF polypeptide is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-20. In further embodiments, the ADNF polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO:2.

[0250] The polypeptides and / or therapeutic agents described herein can be provided to a subject by themselves, or as part of a pharmaceutical composition in admixture with a pharma- ceutically acceptable carrier.

[0251] The agents, polypeptides and components of some embodiments of the invention described hereinabove may be included in an article of manufacture, preferably with appropriate instructions for use and labeling indicating, for example, FDA approval for use in treating the indications described herein.

[0252] Such an article of manufacture may, for example, include at least one container containing at least one of the above-described polypeptides and ingredients (e.g., cannabinoids, ketamine, cytokines, etc.) packaged in another container. The article of manufacture may also include suitable buffers and preservatives to improve the shelf life of the article of manufacture.

[0253] As used herein, a "pharmaceutical composition" refers to a preparation of one or more active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0254] As used herein, the term "active ingredient" refers to a polypeptide or therapeutic agent responsible for a biological effect.

[0255] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutical acceptable carrier" may be used interchangeably and refer to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the compound being administered. Included in these terms are adjuvants.

[0256] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0257] Techniques for drug formulation and administration can be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, incorporated herein by reference.

[0258] Suitable routes of administration may include, for example, oral, sublingual, topical, intradermal, rectal, transmucosal (including eye drops), particularly nasal, intestinal or parenteral delivery (including intramuscular, subcutaneous, transdermal (by pressure) and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac (e.g., into the right or left ventricular cavity), injection into a common coronary artery, intravenous, intraperitoneal, intranasal, intrapulmonary or intraocular injection).

[0259] According to certain embodiments, the active ingredient is provided systemically.

[0260] According to certain embodiments, the route of administration is intranasal or intrapulmonary.

[0261] According to certain embodiments, the polypeptides are formulated for nasal administration, as described in PCT International Application Publication No. WO 16 / 073,199, the contents of which are incorporated herein by reference in their entireties.

[0262] According to other particular embodiments, the route of administration is intradermal. Methods of administering an active agent to the skin are well known in the art and include, for example, intradermal injections, gels, liquid sprays, devices and patches that contain the active agent and are applied to the external surface of the skin.

[0263] According to some embodiments of the present invention, administration of the active agent to the skin of the subject is performed topically (on the skin).

[0264] According to some embodiments of the invention, administration of an active agent to the skin of a subject is performed non-invasively, for example, using a gel, liquid spray or patch (e.g., reservoir-type and matrix-type patches), or a device containing the active ingredient, that is applied onto the skin of the subject.

[0265] It should be noted that to increase delivery of the active agent to the skin, the active agent may be formulated with a variety of vehicles designed to increase delivery to the epidermal or dermal layers, including, but not limited to, liposomes, dendrimers, noisomes, transfersomes, microemulsions, and solid lipid nanoparticles.

[0266] According to some embodiments of the invention, administration is performed by intradermal injection.

[0267] Traditional approaches for drug delivery to the Central Nervous System (CNS) include neurosurgical strategies (e.g., intrahippocampal (IH), intracranial (IC), intracerebral injection, intracerebroventricular (ICV) injection or infusion or intrathecal administration), molecular manipulation of the agent that attempts to exploit one of the endogenous transport pathways of the BBB (e.g., production of chimeric fusion proteins containing a transport peptide with affinity for an endothelial cell surface molecule in combination with an agent that cannot itself cross the BBB), pharmacological strategies designed to increase the lipid solubility of the agent (e.g., conjugation of a water-soluble agent to a lipid or cholesterol carrier), and temporary disruption of the integrity of the BBB by hyperosmotic disruption (resulting from infusion of a mannitol solution into the carotid artery or use of a biologically active agent such as angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with invasive surgical procedures, size restrictions imposed by inherent limitations of endogenous transport systems, potentially undesirable biological side effects associated with systemic administration of chimeric molecules composed of carrier motifs that may be active outside the CNS, and the possible risk of brain damage in regions of the brain where the BBB is disrupted, which make them suboptimal delivery methods.

[0268] Alternatively, the pharmaceutical composition may be administered in a local rather than systemic manner, for example, by injecting the pharmaceutical composition directly into the tissue area of ​​the patient.

[0269] Pharmaceutical compositions of some embodiments of the present invention may be manufactured by processes well known in the art, e.g., by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0270] Thus, pharmaceutical compositions for use in accordance with some embodiments of the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries, which facilitate processing of the active ingredients into preparations that can be used as medicaments. Appropriate formulations depend on the route of administration selected.

[0271] For injection, the active ingredient of the pharmaceutical composition may be formulated in aqueous solution, preferably in physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological saline buffer, or physiological saline or slow release solution. For transmucosal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art.

[0272] For oral administration, pharmaceutical compositions can be easily formulated by combining the active compounds with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by patients. Pharmacological preparations for oral use can be made using solid excipients, and the resulting mixture can be milled if necessary, and the mixture of granules can be processed to obtain tablets or dragee cores, after adding suitable auxiliaries as necessary. Suitable excipients are, in particular, sugars including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carbomethylcellulose, and / or fillers such as physiologically acceptable polymers, such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0273] According to certain embodiments, the pharmaceutical composition is formulated for oral administration.

[0274] The dragee core is provided with a suitable coating.For this purpose, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and concentrated sugar solutions which may optionally contain suitable organic solvents or solvent mixtures may be used.Dyes or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0275] Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, as well as soft sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Stabilizers may also be added. All formulations for oral administration should be in dosages suitable for the selected route of administration.

[0276] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0277] According to certain embodiments, the pharmaceutical compositions are formulated for inhalation (eg, intranasally or intrapulmonary).

[0278] For administration by inhalation, the active ingredient for use according to some embodiments of the present invention is conveniently delivered in the form of an aerosol spray from an overpressurized pack or nebulizer by using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in a dispenser may be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0279] Pharmaceutical compositions suitable for use in some embodiments of the present invention for intranasal administration are conveniently delivered as described in U.S. Pat. No. 10,912,819, inventor I. Gozes, the contents of which are incorporated herein by reference in their entireties.

[0280] The pharmaceutical compositions described herein may be formulated for parenteral administration, for example, by bolus injection or continuous infusion. The formulations for injection may be provided in unit dosage form, for example, in ampoules or multi-dose containers, optionally with the addition of preservatives. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Slow-release formulations may also be used in the preparation of pharmaceutical compositions for parenteral administration.

[0281] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Furthermore, suspensions of the active ingredient may be prepared as suitable oily or aqueous injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. If desired, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredient to allow the preparation of highly concentrated solutions.

[0282] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free aqueous solution, before use.

[0283] Pharmaceutical compositions of some embodiments of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[0284] The pharmaceutical compositions of some embodiments of the present invention may also be formulated for sustained release to enhance serum half-life. Such sustained release systems are well known to those skilled in the art and include, for example, microcapsules and nanoparticles. According to certain embodiments, the ProLease biodegradable microsphere delivery system for proteins and peptides (Tracy, 1998, Biotechnol. Prog, 14, 108; Johnson et al., 1996, Nature Med. 2, 795; Herbert et al., 1998, Pharmaceut. Res. 15, 357) is a dry powder composed of biodegradable polymeric microspheres containing proteins in a polymer matrix that can be formulated as a dry formulation with or without other agents.

[0285] Pharmaceutical compositions suitable for use in connection with some embodiments of the present invention include compositions in which the active ingredient is contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount refers to an amount of active ingredient effective to prevent, reduce, or ameliorate the symptoms of a disease (e.g., ARDS, an infection, e.g., a coronavirus infection) or to prolong the survival of a subject receiving treatment.

[0286] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0287] For any preparation used in the method of the present invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine a useful dose in humans.

[0288] Toxicity and therapeutic efficacy of the active ingredients described herein may be determined by standard formulation procedures in vitro in cell cultures or experimental animals. Data obtained from these in vitro and cell culture assays and animal studies may be used in formulating various dosages for use in humans. Dosages may vary depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage may be chosen by the individual physician in view of the patient's condition. (See, for example, Fingl et al., (1975), "The Pharmacological Basis of Therapeutics", Ch.1, p.1.)

[0289] For a complete toxicity evaluation of the ADNP polypeptide of SEQ ID NO:2, see Gozes I. Front Neurol. 2020 Nov. 24, 11:608444, the contents of which are incorporated by reference in their entirety into this specification.

[0290] Dosage amount and interval may be adjusted individually to provide a level of active ingredient sufficient to induce or suppress the biological effect (Minimal Effective Concentration (MEC)). The MEC varies for each preparation but can be estimated from in vitro data. Dosages necessary to achieve the MEC depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0291] Doses determined in mouse models can be translated for treatment of other species (e.g., humans and other animals diagnosed with the disease). An FDA-approved conversion table is provided in Reagan-Shaw S. et al., FASEB J. 22:659-661 (2007).

[0292] The human equivalent dose is calculated as follows: HED (mg / kg) = Animal Dose (mg / kg) × (Animal K m / Human K m ).

[0293] Depending on the severity and responsiveness of the condition being treated, dosing may be a single or multiple administration, and the course of treatment may last from a few days to a few weeks, or until a ameliorative effect occurs or a diminution of pathology is achieved. It will be appreciated that treatment is typically long-term, and in most cases a chronic course of treatment, as the target patient population includes genetically impaired individuals whose neurodegenerative conditions require continued attention.

[0294] According to certain embodiments, the polypeptide is administered once or twice daily.

[0295] The amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0296] According to some embodiments of the invention, the polypeptide is provided in an amount ranging from 0.0001 mg / kg to 1,000 mg / kg, including any intermediate subranges and values ​​therebetween, such as 0.001 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 15 mg / kg, 50 mg / kg, or 500 mg / kg per dose. According to certain embodiments, the polypeptide is provided in an amount ranging from 0.05 to 0.1 mg / kg, such as 0.08 mg / kg. In more specific embodiments, the polypeptide is provided in an amount ranging from 0.01 mg / kg body weight to 2 mg / kg body weight, including any intermediate subranges and values ​​therebetween, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 129, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 44, 45, 46, 47, 48, 49 or 50 mg / 70 kg of subject body weight. In a still further embodiment the polypeptide is provided in an amount in the range of 1 to 40 mg / 70 kg of subject's body weight, in particular 5, 15 or 30 mg / 70 kg of subject's body weight.

[0297] According to a particular embodiment, the polypeptide is provided in an amount ranging from 0.1 to 1 mg / kg, for example 0.4 mg / kg, administered, for example, subcutaneously.

[0298] According to certain embodiments, the polypeptide is provided, e.g., intranasally, in an amount ranging from 0.05 to 0.5 mg / kg, e.g., 0.2 mg / kg (15 mg for a 70 kg subject) or 0.07 mg / kg (5 mg for a 70 kg subject).

[0299] The compositions of some embodiments of the present invention may be provided in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also correspond to a notice associated with the container in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the composition or form of administration in human or veterinary medicine. Such notice may, for example, be a label approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product insert. The compositions comprising the preparations of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for the treatment of an indicated condition, as further detailed above.

[0300] As used herein, the term "about" refers to ±10%.

[0301] The terms "comprises," "comprising," "includes," "including," "having" and variations thereof mean "including but not limited to."

[0302] The term "consisting of" means "including and limited to."

[0303] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0304] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0305] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0306] Whenever a numerical range is given herein, it is meant to include any recited numbers (fractional or integer) within the given range. The terms "ranging" between a first denoted number and a second denoted number and "ranging" from a first denoted number to a second denoted number are used interchangeably herein and are meant to include the first denoted number and the second denoted number, and all fractional and integer numbers therebetween.

[0307] As used herein, the term "method" refers to manner, means, techniques, and procedures for accomplishing a given task, and includes, but is not limited to, manner, means, techniques, and procedures that are well known or readily developed from known manner, means, techniques, and procedures by practitioners in the chemical, pharmaceutical, biological, biochemical, and medical fields.

[0308] As used herein, the term "treating" includes arresting, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating a clinical or cosmetic symptom of a condition, or substantially preventing the appearance of a clinical or cosmetic symptom of a condition.

[0309] When referring to a particular sequence listing, such reference should also be understood to encompass sequences that substantially correspond to their complementary sequences, including minor sequence variations resulting from, for example, sequencing errors, cloning errors, or other changes resulting in base substitutions, deletions, or additions, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively less than 1 in 100 nucleotides, alternatively less than 1 in 200 nucleotides, alternatively less than 1 in 500 nucleotides, alternatively less than 1 in 1000 nucleotides, alternatively less than 1 in 5,000 nucleotides, alternatively less than 1 in 10,000 nucleotides.

[0310] It is understood that certain features of the invention that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention are described in the context of a single embodiment for brevity, but may also be provided separately or in any suitable subcombination or as desired in other described embodiments of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment cannot function without those elements.

[0311] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below are found to be experimentally supported in the following examples. EXAMPLES

[0312] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0313] In general, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully explained in the literature, see, e.g., Molecular Cloning: A laboratory Manual, Sambrook et al. (1989), Current Protocols in Molecular Biology, Volumes I-III Ausubel, RM, ed. (1994), Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Baltimore, Maryland (1989), Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988), Watson et al., Recombinant DNA, Scientific American Books, New York, Birren et al. (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. New York (1998), U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057; Cell Biology: A Laboratory Handbook, Volumes I-III Cellis, JEed. (1994); Culture of Animal Cells-A Manual of Basic Technique by Freshney, Wiley-Liss, NY (1994), Third Edition; Current Protocols in Immunology, Volumes I-III Coligan JEed.(1994), Stites et al. (eds), Basic and Clinical Immunology (8th Edition), Appleton & Lange, Norwalk, CT (1994), Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York, NY (1994). York (1980); available immunoassays are widely described in the patent and scientific literature, see, e.g., U.S. Pat. Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771, and 5,281,521; "Oligonucleotide Immunoassays" in US Pat. Synthesis" Gait, MJ, ed. (1984), "Nucleic Acid Hybridization" Hames, BD, and Higgins SJ, eds. (1985), "Transcription and Translation" Hames, BD, and Higgins SJ, eds. (1984), "Animal Cell Culture" Freshney, RI, ed. (1986), "Immobilized Cells and Enzymes" IRL Press, (1986), "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol.1-317, Academic Press, "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization--A Laboratory Course Manual", CSHL Press (1996), all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated by reference herein.

[0314] Materials and Methods Cell culture - Human neuroblastoma SH-SY5Y cells differentiated into neuronal-like cells (10 μM retinoic acid) 15 , human embryonic kidney 293 (HEK293) cell line, and induced pluripotent stem cells (iPSCs) differentiated into neural cells. 37 were subjected to immunohistochemistry and / or co-immunoprecipitation.

[0315] Docking of SIRT1 to the EB1 homodimer via the SXIP motif Available structures: 1.SIRT1:PDB ID 4IG9 38 X-ray structure of the open state of SIRT1. Chain A was used. 2. EB1 homodimer: 3TQ7 as disclosed 39 and 3GJO 40 Made using 12 .

[0316] Docking Method: 1.Patchdock 41 was used to dock SIRT1 into the EB1 homodimer using the known binding sites (residues 452-456 of SIRT1 and residues 219, 220 and 223 of EB1). 2. Firedock 42、43 The Patchdock results were refined using the full refinement protocol. 3. Rosetta Dock 44 The best seven docking poses from Firedock were further refined and scored using a high-resolution docking protocol using .

[0317] Co-immunoprecipitation assay - Proteins were extracted from differentiated human neuroblastoma SH-SY5Y cells using lysis buffer (Pierce, Rockford, IL) and immunoblotted as previously described. 13、25 , and subjected to Co-IP analysis according to the manufacture's protocol. Briefly, 10 μg of the antibody of interest (EB1, ab53358 EB3, ab99287 Abcam, Cambridge, UK) was cross-linked to 30 μl of A / G PLUS-agarose beads (provided by the Co-IP kit). 1 mg of protein extract was incubated overnight at 4 °C in an end-over-end shaker (ROTAMIX RM1, Riga, Latvia). To enhance the interaction with EB1 protein ...) as described. 13、25After 2 min, 3 mg of NAP (SEQ ID NO:2) was added. The immunoprecipitated material was then examined by western blot analysis using primary antibodies as follows: mouse monoclonal SIRT1 antibody (1:1000, ab110304, Cambridge, UK), mouse monoclonal ADNP F9 (1:200, SC-376674, Santa Cruz, CA), rat monoclonal EB1 (1:500), goat polyclonal EB3 (1:2500) and rabbit monoclonal tau (1:2500, ab32057, Abcam). Proteins were then visualized with SuperSignal Chemiluminescent Substrates (Thermo Scientific, Rockford, IL) using horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (1:5000, Jackson, Hamburg, Germany), goat anti-rat (1:5000, Jackson), donkey anti-goat (1:5000, Jackson), and goat anti-rabbit (1:5000, Abcam).

[0318] The RNA Sequencing Single Cell Analysis-NCBI website was searched for datasets derived from single-cell transcriptomes of the developing human brain. 1) Single-cell RNA sequencing data from human cortical specimens (48 samples from 5.85–37 weeks postconception, PRJNA295469) 45 ) were analyzed using the UCSC Cell Browser (www(dot)cells(dot)ucsc(dot)edu / ?ds=cortex-dev). Single-cell 2D plots were visualized by the t-SNE algorithm, and ADNP- and SIRT1-expressing cells were labeled with black circles. 2) Single-cell RNA sequencing data from 124 cells derived from human embryonic stem cells and human preimplantation embryos (GSE36552) 46 The single-cell expression levels of ADNP, SIRT1, MAPR1, and MAPR3 were analyzed using the Single Cell Expression Atlas. 47 The analysis was performed using and t-SNE plots were generated with perplexity=50.

[0319] Tissue RNA expression analysis - Total bulk RNA expression levels of ADNP, SIRT1, MAPR1, and MAPR3 in human tissues were analyzed using Genotype-Tissue Expression (GTEx) 48 Data were analyzed using t-SNE. Expression levels were visualized using UCSC Cell Browser (www(dot)cells(dot)ucsc(dot)edu / ?ds=gtex8).

[0320] Animals - All animal procedures were approved by the Animal Care and Use Committee of Tel Aviv University and the Israeli Ministry of Health. 11、12 Adnp on a mixed C57BL and 129 / Sv background + / - Mice were cultured as previously described. 1、16、49 For continuous breeding, the ICR outbred mouse strain was used. 12、49 Animals were housed on a 12-h light / 12-h dark cycle and provided rodent chow and water ad libitum. Genotyping was performed by Transnetyx (Memphis, Tenn.).

[0321] Histone modification screening - Histone extraction was performed on 3- and 5-month-old male and female AdnpT cells using a Histone Extraction Kit (ab113476, Abcam) according to the manufacturer's protocol. + / - and Adnp + / + Histone proteins were extracted from mouse hippocampus. Histone proteins were measured by Bradford Protein Assay (Cat. No. 500-0006, BIO-RAD). Histone H3 Modification Multiplex Assay Kit (ab185910, Abcam) was used to screen and quantify histone H3 modifications in Adnp mice. 500ng of total histone protein was used per assay according to the protocol booklet. Absorbance was measured in a microplate reader at a wavelength of 450nm with a reference wavelength of 655nm.

[0322] Motif Analysis - Factorbook 50 The database was compiled using the ENCODE chip-seq dataset. 51 The binding sites from ENCSR000AQG, ENCSR000AQG, SMARCA4, ENCSR000EHO, and YY1 in K562 cells were analyzed. Chip-seq data binding peaks were analyzed to generate enrichment of motif sequences. The average histone modification profile was calculated using Factorbook 50 The ADNP chip-seq binding site database was used to generate histone peaks that were proximal (<1 kb) to the transcription start site (TSS) and distal to the TSS, each of which accounted for the majority of sequence reads. 50 The region surrounding the position having the .alpha.-amino acid sequence is 2 kb.

[0323] Chip-seq Data Mining - Cistrome DB 52 for Chip-seq data mining and the WashU epigenome browser 32 Chip-seq binding peaks for H3K79me2 (GSM733653), ADNP (eGFP-ADNP tagged K562 cell line, GSE105573), HDAC2 (GSM1003447), YY1 (GSM803470, GSM803446), and SMARCA4 (GSE91946) from erythroblasts (cell line K562) were downloaded and further visualized in the WashU epigenome browser.

[0324] Correlation matrix analysis - Human tissue RNA expression levels were analyzed in 95 human individuals 53RNA-seq datasets from 27 normal tissue samples from the NCBI Gene Expression Omnibus (GEO) GEO website were screened for datasets derived from human AD postmortem brain tissue or blood from living patients and controls. An AD postmortem brain dataset (GSE5281) from the entorhinal cortex, hippocampus, medial temporal gyrus, posterior cingulate gyrus, superior frontal gyrus, and primary visual cortex, including samples from 74 controls and 87 patients. 53 The second cohort analyzed was a blood dataset (GSE63060) containing samples from 105 controls and 146 AD patients. 54 In addition, the BrainSpan atlas of postmortem brain structure spanning human brain development 51 We evaluated RNA-seq data from

[0325] For the Pearson correlation of the RNA expression levels of Sirt1 and Adnp, Adnp + / - and Adnp + / + Mouse, mouse hippocampus (N=23) RNA-seq dataset (GSE72664) 12 Expression levels were downloaded and Pearson correlation coefficients were calculated using R software. Correlation matrices were generated using the Corrplot package, with asterisks indicating significance levels. *** P<0.001, ** P < 0.01, ** P<0.05.

[0326] Further details can be found in Hadar A, Kapitansky O et al., Mol Psychiatry. 2021 November, 26(11):6550-6561, the entire contents of which are incorporated herein by reference.

[0327] Example 1 ADNP and SIRT1 share a microtubule end-binding protein (EB1) motif and interact with EB1 / EB3 at the single cell and biochemical levels STRING protein interaction analysis (www(dot)string-db(dot)org / ) showed the complexity of ADNP interacting and SIRT1 network proteins (data not shown). However, this current knowledge of potential ADNP-SIRT1 interactions does not imply any major focus. Nevertheless, TP53 appears in the SIRT1 network and, as previously shown, TP53 is regulated by ADNP. 4 , suggesting a common pathway.

[0328] Therefore, we sought to discover whether there are potential physical interaction points between ADNP and SIRT1, possibly through other protein mediators. To this end, we used Eukaryotic Linear Motif (ELM) prediction analysis. 55 Using chromatin analysis (data not shown), we uncovered multiple binding motifs for WD-repeat-containing protein 5 (WDR5), which mediates the assembly of histone-modifying complexes (Fig. 1A). As a scaffolding protein, WDR5 contributes to histone modifications. By recruiting the core histone methylation and acetylation complexes, WDR5 mediates H3K4 methylation / demethylation. 56 and H4 acetylation on several lysine residues 57 plays an important role in

[0329] Subsequently, the inventors demonstrated LC3 interaction sites in both ADNP as SIRT1, as well as the eukaryotic initiation factor 4E interaction site. 49 (FIG. 1A). Furthermore, the SIRT1-EB binding domain was demonstrated together with the SSIP at SIRT1 positions 453-456 (FIG. 1A) as well as with the well-known ADNP SIP motif.

[0330] SKIP motif from ADNP 12 and NAPVSIPQ motif 58Docking in silicon for SIRT1 was previously shown. Looking at the SIRT1-EB1 interaction, 88 docking poses were obtained from Patchdock and refined with Firedock. The 7 best docking poses were then further refined using the Rosetta Dock high resolution application. 1000 decoys were calculated for each docking pose. The best pose was selected by the total score of the complex (Figure 1B).

[0331] Considering the shared interacting proteins for ADNP and SIRT1, the physical interaction was further addressed experimentally at the cellular level by immunocytochemistry and at the protein biochemical level by co-immunoprecipitation. Specifically, HEK293T cells were immunostained with antibodies recognizing ADNP or SIRT1 and cell nuclei were counterstained / visualized with DAPI. Nuclear co-localization of the two proteins is represented in yellow and quantified on the graph (Figure 1C). The biological significance of cellular co-localization was extended by staining human iPSC-derived neuronal cells. 37 (Fig. 1D), revealing colocalization between the nucleus and cytoplasm.

[0332] Next, we investigated the ADNP expression in SH-SY5Y neuroblastoma cells, which are mainly cytoplasmic. 9 We performed co-immunoprecipitation with proteins extracted from human iPSC-derived neural progenitor cells. The results (Figure 1E) showed specific co-immunoprecipitation of ADNP and SIRT1 in the presence of either EB1 or EB3 antibodies. We also used extracts from human iPSC-derived neural progenitor cells to enhance the ADNP-EB-tau interaction. 13 Co-immunoprecipitation with EB1 was performed in the absence (Fig. 1F) and presence (Fig. 1G) of the ADNP fragment NAP (tau binds directly to SIRT1). 23It is known that SIRT1 is a marker for tau activation in EB1 antibody-eluted cells. The results did not reveal SIRT1 in the EB1 antibody-eluted fractions (Fig. 1F, upper panel, E1-E3). Furthermore, the load was saturated and EB1-like immunoreactivity was also detected in the flow-through (Fig. 1F, FT, lower panel). The FT fraction was then reacted with immobilized EB1 antibody containing 3 mg of NAP (SEQ ID NO:2). Electrophoresis and immunoblotting then identified a faint SIRT1-like band at the expected position for the SIRT1 antibody, and a potential tau band (Fig. 1G, arrow) that was more strongly identified by the tau antibody (lower panel).

[0333] Genomic information processing tools and publicly available single-cell RNA-seq libraries 45 Utilizing , we further extended the colocalization question to the developing human cortex, revealing 16.8% of cells co-expressing ADNP and SIRT1 (Figure 2A). Looking at cellular expression in multiple human tissues, we found high similarity in the patterns of ADNP and SIRT1 expression (Figure 2B). Interestingly, when looking at EB1 and EB3 (mRNA, MAPRE1 and MAPRE3) expression (data not shown), MAPRE1, but not MAPRE3, mimicked the ADNP / SIRT1 expression pattern. This was also reproducible in stem cells (Figure 2C) and during development (data not shown).

[0334] Further details can be found in Hadar A, Kapitansky O et al., Mol Psychiatry. 2021 November, 26(11):6550-6561, the entire contents of which are incorporated herein by reference.

[0335] Example 2 ADNP and SIRT1 are transcriptionally co-regulated and both regulate specific histone H3 modifications SWI / SNF complex containing ADNP 5 chromatin remodeling complex 33 SIRT1 is involved in chromatin remodeling associated with oocyte aging and exhibits changes in histone methylation. 59Histone H3 methylation has also been associated with ADNP, HP1 (binding to ADNP) 2 , was found to recruit ADNP to H3K9me3-marked pericentromeric heterochromatin for silencing of the major satellite repeats. 6 Furthermore, loss of ADNP resulted in an increase in the H3K4me3 / H3K27me3 ratio at key primitive endoderm gene promoters in embryonic stem cells. 8 .

[0336] Subsequently, using a comprehensive approach, we identified Adnp1, which shows significant dysregulation of gene expression at the RNA-seq level. + / + Adnp the mouse + / - We investigated Adnp-regulated histone modifications in the hippocampus of 5-month-old females compared with mice. 12 Although no Adnp gene dosage effect was observed on the level of total histone H3 methylation (Figure 3A), specific methylation sites were affected. For example, H3K79me2 (red dotted box) was identified as significantly decreased due to Adnp deletion. To substantiate these results, we further investigated the effect of Adnp gene dosage on the level of total histone H3 methylation (Figure 3B). 63 , and SIRTs that regulate red blood cell maturation and globin expression 61 In the 1990s and 2000s, ADNP, SIRT1, and MAPR1 (which co-localize in the same cells, Figures 1C-G and 2A-C) were expressed by H3K79me2 (GSM733653), which regulates their unique expression, and ADNP (GSE105573). 2、60 , HDAC2 (GSM1003447) and YY1 (GSM803470, GSM803446) interact with SIRT1 61、62 , ADNP interacts with BRG1 (SMARCA4) (GSE91946) 5We investigated Chip-seq binding peaks on promoter / regulatory regions (data not shown) of ADNP. WashU epigenome browser identified adjacent histone modification peaks of major ADNP-related modifications, most prominently in activated and transcriptional histone marks (Figure 3B box). Figure 3B further shows highly reproducible promoter binding of all proteins tested, suggesting co-interaction and co-regulation at the transcriptional level. Mechanistically, by exploring promoter regions, a motif shared between ADNP, YY1, BRG1 (SMARCA4) and HDAC2 was identified, with HDAC2 showing the highest similarity to ADNP (Figure 3C). In further mechanistic exploration, key histone interacting proteins were selected, suggesting a novel transcriptional regulatory / chromatin modification complex (Figure 4A). Further studies also analyzed sex- and age-dependent histone H3 methylation, showing distinct potential hotspot patterns of methylation, suggesting complex regulation (data not shown).

[0337] Further details can be found in Hadar A, Kapitansky O et al., Mol Psychiatry. 2021 November, 26(11):6550-6561, the entire contents of which are incorporated herein by reference.

[0338] Example 3 Correlation of expression and developmental coregulation shows striking dysregulation in postmortem brains of Alzheimer's disease Since the colocalization and interaction of ADNP, SIRT1 and MAPR1 (EB1) was shown (Figure 1A-G and Figure 2A-C), it was further interesting to investigate the expression correlation of ADNP and SIRT1 and related gene products. Figure 4B shows a correlation matrix plot showing the RNA-seq expression levels of 27 normal tissues from 95 human individuals (data from BioProject: PRJEB4337). 53), showing a very high correlation between SIRT1 and ADNP. Figure 4C extends this analysis to selected RNA species encoding ADNP-SIRT1 interacting proteins in normal brain. High correlation was shown except for HAT1, RBBP7, HDAC1, and MAPRE3, and most transcripts showed positive correlation except for MAPRE3 and NMNAT1.

[0339] To confirm the correlation results, two additional experimental paradigms were used: 1) Adnp + / - Adnp + / + Mouse evaluation 11 and 2) using additional publicly available human datasets. Mouse results are shown in FIG. 4B (inset, RNA-seq). Further analysis of males and females separately revealed extensive correlation in 5-month-old females (data not shown, r=1, p=0.005), but not in males or 1-month-old mice. Confirmation by qRT-PCR using twice the number of mice at 1 month of age showed a correlation in males (data not shown, r=0.669, p=0.0343), suggesting possible age- and sex-dependence.

[0340] For humans, the classified AD and ASD blood samples showed little difference between the AD and ASD data and the respective control data (data not shown). 64 was evaluated and a correlation matrix plot of postmortem brain gene expression levels of grouped AD patients and controls is shown (Figure 5). Positive correlations are shown in blue scale and negative correlations in red scale. Dramatic differences were observed, with a complete loss of correlation in AD brains (Figure 5). By comparing different brain regions, highly significant differences in the primary visual cortex and superior frontal gyrus, related to visuospatial cognition, were identified. 65 ). In Parkinson's disease brains compared with controls, the correlations remained, except in the most affected area, the substantia nigra (data not shown).

[0341] FIG. 6 summarizes the findings presented in Examples 1-3 above, modeling multiple non-limiting SIRT1-ADNP interactions at the chromatin and cytoplasmic levels.

[0342] Further details can be found in Hadar A, Kapitansky O et al., Mol Psychiatry. 2021 November, 26(11):6550-6561, the entire contents of which are incorporated herein by reference.

[0343] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0344] It is the intention of the applicant(s) that all publications, patents, and patent applications mentioned herein be incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. Furthermore, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. Additionally, any priority document(s) of this application are incorporated by reference herein in their entirety.

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Claims

1. an ADNF polypeptide as an active ingredient, which has neurotrophic / neuroprotective activity in an in vitro cortical neuronal cell culture assay; 1. An article of manufacture comprising: (i) a SIRT1 activator; (ii) an anti-aging agent that is not an antioxidant; (iii) an immunomodulatory agent selected from the group consisting of a chemokine receptor modulator, an immune checkpoint modulator, and a cytokine, wherein the cytokine is not IFNβ; (iv) bumetanide or an analog or derivative thereof; (v) a cannabinoid; and (vi) ketamine or an analog or derivative thereof.

2. The article of manufacture of claim 1, wherein the article is characterized by at least one of the following: (i) the polypeptide and the SIRT1 activator are provided in a co-formulation or in separate formulations; (ii) the SIRT1 activator is a small molecule; (iii) the anti-aging agent is not an antioxidant; (iv) the cytokine is not IFNβ; (v) the chemokine receptor is selected from CCR5 and CXCR4; (vi) the regulator is an inhibitor; (vii) the chemokine receptor modulator is selected from the group consisting of maraviroc, leronlimab, aplaviroc, vicriviroc, plerixafor, mavorixafor, BL-8040, and TGO-0054, or an analogue or derivative thereof; (viii) the cytokine is selected from the group consisting of IL-6, IL-10, and TNFα; (ix) the cannabinoid is selected from the group consisting of THC and CBD.

3. The article or method of manufacture of claim 2, characterized by one of the following: (i) the SIRT1 activator is selected from the group consisting of NAD+ or an analog or derivative thereof, or Nicotinamide Riboside (NR), resveratrol, quercetin, butein, beverine, curcumin, fisetin, honokiol, YK3-237, SRT1720, SRT1460, SRT2183, STAC-5, STAC-9, STAC-10, BML-278, and piceatannol, or an analog or derivative thereof; (ii) the anti-aging agent is selected from the group consisting of rapamycin, metformin, melatonin, carnosine, nicotinamide mononucleotide, δ-sleep inducing peptide and low molecular weight klotho enhancer, or an analog or derivative thereof, or a calorie restricted diet; (iii) the anti-aging agent is a SIRT1 activator.

4. The article of manufacture described in claim 1, wherein the ADNF polypeptide is capable of binding to EB1 and / or EB3.

5. The article of manufacture of claim 1, wherein the ADNF polypeptide is selected from an ADNF III polypeptide and an ADNF I polypeptide.

6. The article of manufacture of claim 5, wherein the ADNF III polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-22, and the ADNF I polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:24-48. (i) the polypeptide is ADNF III having the formula (R1)x-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-(R2)y (SEQ ID NO:49), wherein R1 is an amino acid sequence comprising from 1 to about 40 amino acids, each amino acid being independently selected from the group consisting of naturally occurring amino acids and amino acid analogs, and R2 is an amino acid sequence comprising from 1 to about 40 amino acids, each amino acid being independently selected from the group consisting of naturally occurring amino acids and amino acid analogs, and x and y are independently selected and equal to 0 or 1; 6. The article of manufacture of claim 5, wherein the polypeptide is ADNF I having the formula (R 1 ) x -Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-(R 2 ) y (SEQ ID NO:50), or an analog thereof, where R 1 is an amino acid sequence comprising from 1 to about 40 amino acids, each amino acid being independently selected from the group consisting of naturally occurring amino acids and amino acid analogs, and R 2 is an amino acid sequence comprising from 1 to about 40 amino acids, each amino acid being independently selected from the group consisting of naturally occurring amino acids and amino acid analogs, and x and y are independently selected and equal to 0 or 1.

8. The article of manufacture of claim 4, wherein the polypeptide is characterized by at least one of the following: (i) the polypeptide comprises at least one D-amino acid; (ii) the polypeptide is less than 50 amino acids in length, or less than 20 amino acids in length; (iii) the polypeptide is linked to a cell-permeable or stabilizing moiety.

9. 1. An active ingredient for use in a method of treating a disease selected from the group consisting of ADNP syndrome, Dravet syndrome, Fragile X syndrome, SYNGAP1 associated intellectual disability, Phelan-McDermid syndrome, GRIN disorder, CHD8 associated disorder, DYRK1A syndrome, POGZ syndrome, FOXP1 syndrome, SLC5A1 associated disorder, Coffin-Siris syndrome, ARID1B associated syndrome, KMT5B syndrome, PTEN autism syndrome, Rett syndrome, Okihiro syndrome plus developmental delay, Angelman syndrome, Noonan syndrome, Kleefstra syndrome, and Smith-McGinnis syndrome, wherein said active ingredient is selected from a SIRT1 activator, an anti-aging agent, and an immunomodulator selected from the group consisting of a chemokine receptor modulator, an immune checkpoint modulator, and a cytokine, thereby treating said disease in said subject.

10. The active ingredient for the use according to claim 9, wherein said use is characterized by one of the following: (i) the SIRT1 activator is a small molecule; (ii) the anti-aging agent is not an antioxidant; (iii) the cytokine is not IFNβ; (iv) the chemokine receptor is selected from CCR5 and CXCR4; (v) the modulator is an inhibitor; (vi) the chemokine receptor modulator is selected from the group consisting of maraviroc, leronlimab, aplaviroc, vicriviroc, plerixafor, mavorixafor, BL-8040, and TGO-0054, or an analogue or derivative thereof; (vii) the cytokine is selected from the group consisting of IL-6, IL-10, and TNFα.

11. The active ingredient for use according to claim 10, wherein said use is characterized by one of the following: (i) the SIRT1 activator is selected from the group consisting of NAD+ or an analog or derivative thereof, or Nicotinamide Riboside (NR), resveratrol, quercetin, butein, beverine, curcumin, fisetin, honokiol, YK3-237, SRT1720, SRT1460, SRT2183, STAC-5, STAC-9, STAC-10, BML-278, and piceatannol, or an analog or derivative thereof; (ii) the anti-aging agent is selected from the group consisting of rapamycin, metformin, melatonin, carnosine, nicotinamide mononucleotide, δ-sleep inducing peptide and low molecular weight klotho enhancer, or an analog or derivative thereof, or a calorie restricted diet; (iii) the anti-aging agent is a SIRT1 activator.

12. An active ingredient for use according to any one of claims 9 to 11, wherein the use further comprises administering to the subject a therapeutically effective amount of an ADNF polypeptide having neurotrophic / neuroprotective activity in an in vitro cortical neuron culture assay.

13. An active ingredient for use according to claim 12, wherein the ADNF polypeptide is capable of binding to EB1 and / or EB3.

14. The method of claim 12, wherein the ADNF polypeptide is selected from an ADNF III polypeptide and an ADNF I polypeptide.

15. The method of claim 14, wherein the ADNF III polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-22 and the ADNF I polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-48.

16. The polypeptide of claim 1, wherein the polypeptide is ADNF III having the formula (R1)x-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-(R2)y (SEQ ID NO:49), wherein R1 is an amino acid sequence comprising from 1 to about 40 amino acids, each amino acid being independently selected from the group consisting of naturally occurring amino acids and amino acid analogs, and R2 is an amino acid sequence comprising from 1 to about 40 amino acids, each amino acid being independently selected from the group consisting of naturally occurring amino acids and amino acid analogs, and x and y are independently selected and equal to 0 or 1; 15. The method of claim 14, wherein the polypeptide is ADNF I having the formula (R1)x-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-(R2)y (SEQ ID NO:50), or an analog thereof, wherein R1 is an amino acid sequence comprising from 1 to about 40 amino acids, each amino acid being independently selected from the group consisting of naturally occurring amino acids and amino acid analogs, and R2 is an amino acid sequence comprising from 1 to about 40 amino acids, each amino acid being independently selected from the group consisting of naturally occurring amino acids and amino acid analogs, and x and y are independently selected and equal to 0 or 1.

17. The active ingredient for use according to any one of claims 9 to 16, wherein the polypeptide is characterized by at least one of the following: (i) the polypeptide comprises at least one D-amino acid; (ii) the polypeptide is less than 50 amino acids in length, or less than 20 amino acids in length; (iii) the polypeptide is linked to a cell-permeable or stabilizing moiety.

18. An active ingredient for the use according to claim 9, characterized by at least one of the following: (i) the subject is female or male; (ii) the subject is under 18 years of age or over 60 years of age.

19. The method for use according to claim 9, characterized in that the active ingredient is at least one of the following: (i) the disease is associated with aging; (ii) the disease is an inflammatory disease; (iii) the disease is a neurodegenerative disease or a cognitive disorder; (iv) the disease is autism spectrum disorder and / or intellectual disability; (v) The disease is ADNP syndrome; (vi) the disease is selected from the group consisting of stress, anxiety, bipolar disorder, schizophrenia, and aggression; (vii) the disease is selected from the group consisting of hypertension, swelling, congestive heart failure, liver disease and kidney disease.

20. The active ingredient for use according to claim 19, wherein the disease is a neurodegenerative disease or cognitive disorder selected from mild cognitive impairment, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), Progressive Supranuclear Palsy (PSP), and Alzheimer's disease.

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